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PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/156495 Please be advised that this information was generated on 2017-06-12 and may be subject to change. Autosomal recessive hearing impairment Hearing of & listening to patients Anne M.M. Oonk Autosomal recessive hearing impairment Hearing of & listening to patients Anne M.M. Oonk 2016 ISBN 978-90-9029535-0 Design/lay-out Promotie In Zicht, Arnhem Print Ipskamp Printing, Enschede © Anne M.M. Oonk, the Netherlands, 2016 All rights reserved. No parts of this thesis may be reproduced or transmitted in any form or by any means, electronically, mechanically, by print of otherwise, without prior written permission of the author. The research presented in this thesis was performed at the department of Otorhinolaryngology and the department of Human Genetics of the Radboud University Medical Center, Nijmegen, the Netherlands with financial support of ZonMW Financial support for the publication of this thesis was provided by: ABN Amro, Daleco Pharma b.v., DOS medical/ kno-winkel.nl, EmID audiologische apparatuur, Specsavers, Meda Pharma, Meditop Medical Products, Cochlear, Phonak, Olympus Nederland B.V., ZEISS, Tramedico. Autosomal recessive hearing impairment Hearing of & listening to patients Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus, volgens besluit van het college van decanen in het openbaar te verdedigen op donderdag 14 april 2016 om 12.30 uur precies door Anne Marthe Maria Oonk geboren op 3 mei 1987 te Haarlem Promotor Prof. Dr. J.M.J. Kremer Copromotor Dr. R.J.E. Pennings Manuscriptcommissie Prof. dr. F.P.M. Cremers (voorzitter) Prof. dr. P. van Dijk (Universitair Medisch Centrum Groningen) Dr. V. Topsakal (Universitair Medisch Centrum Utrecht) Contents Chapter 1 Introduction 1.1 General introduction 1.2 Features of autosomal recessive nonsyndromic hearing impairment; a review to serve as a reference Chapter 2 Novel genotype-phenotype correlations 7 9 29 71 2.1 Similar phenotypes caused by mutations in OTOG and OTOGL 73 2.2 Nonsyndromic hearing loss caused by USH1G mutations: widening the USH1G disease spectrum 93 2.3 Progressive hearing loss and vestibular dysfunction caused by a homozygous nonsense mutation in CLIC5 Chapter 3 Expanding a genotype-phenotype correlation 3.1 Vestibular function and temporal bone imaging in DFNB1 Chapter 4 Psychosocial impact of a genetic diagnosis 4.1 Psychological impact of a genetic diagnosis on hearing impairment - an exploratory study 109 133 135 157 159 Chapter 5 Discussion and conclusion 175 Chapter 6 Summary / Samenvatting 193 6.1 English summary 195 6.2 Nederlandse samenvatting 201 Chapter 7 Dankwoord 207 Chapter 8 Curriculum Vitae 213 Chapter 9 List of publications 217 Chapter 10 List of abbreviations 223 1 Introduction 1.1 General Introduction Introduction | 11 Sounds Hearing is the ability to perceive sound. Sounds can be simple, like beeps or pure tones, but sounds can also be more complex like, for example, speech. Hearing, therefore, is an important aspect of communication. Besides sound detection and speech recognition, sound localization is another important feature of hearing. These three components are important in another function of hearing: detection of danger. Sounds are, basically, waves in a medium. These waves can be described by means of three characteristics: frequency, amplitude and propagation velocity. Humans are capable of hearing frequencies between 20 and 20,000 Hz. The most important sound source for humans is the human voice. Sounds are usually consisting of a combination of frequencies, a frequency spectrum. For speech reception, this frequency spectrum is around 2000 Hz 1. Figure 1 Frequency and intensity of sounds. Decibel Hearing Level (dB HL) Hertz (Hz). This image has been copied by courtesy of Phonak. 12 | Chapter 1 Sound pressure, or the amplitude of the wave, is perceived as loudness of sound. This loudness is defined in decibels (dB) or pascal (Pa). The hearing threshold of a normal hearing individual is 0 dB HL (decibel hearing level (20 µPa)) and the pain threshold is about 130 dB HL (20Pa). Propagation velocity depends on the medium. In the external auditory canal and middle ear this medium is air, in the cochlea this is fluid (endolymph and perilymph). However, sound can also be conducted through a solid medium, like bone. The different characteristics of sound waves are detected and processed by the ear. Anatomy and physiology of the ear The ear can be divided in three parts: 1. External ear 2. Middle ear 3. Inner ear The external ear comprises the auricle and the external auditory canal (EAC). The EAC directs sounds from the auricle to the tympanic membrane. The external ear alters sound wave amplitudes and therewith, provides a mechanism for amplification of different sounds within the frequencies of human speech. The middle ear consists of the tympanic membrane, tympanic cavity, ossicles and associated muscles. The middle ear cavity is connected to the mastoid air-cell system. The malleus, incus and stapes are consecutively located between the tympanic membrane and the cochlea. These three ossicles conduct sound from the tympanic membrane towards the cochlea. The middle ear is especially important in transforming sounds, also known as impedance matching. It converses vibration of air into vibration of fluid. When sound travels from air to fluid, it is partially reflected and a lot of sound energy is lost. Due to two features of the middle ear this loss of energy is restricted: 1. The difference of surface size between the tympanic membrane and the stapes foot plate. The sound, in other words, the pressure, applied to the tympanic membrane is transferred to a surface 17 times as small. 2. Lever mechanism of the ossicles. The malleus - incus construction is a lever, which contributes a factor 1.3 to the transmission of sound 2. Sound enters the cochlea through the mobile footplate of the stapes located in the oval window. The cochlea is shaped like a snail shell. This shell consists of three scalae: the scala vestibuli, scala media and scala tympani. These three scalae are separated by Reissner’s membrane and the basilar membrane. Introduction | 13 Organ of corti Located on the basilar membrane is the organ of Corti. It contains the sensory hair cells of the cochlea, which convert the mechanical stimulus of the travelling sound wave through the cochlea into an electric potential. These sensory cells are organized in one row of inner hair cells and three rows of outer hair cells and are surrounded by supporting cells. The tectorial membrane is a gelatinous membrane covering the apical side of the hair cells. At the basal end of the hair cells the synapses of the afferent and efferent neurons are located. In the modiolus of the cochlea, the spiral ganglion that contains the cell bodies of the auditory neurons is found. These auditory neurons are part of the auditory nerve. At the apical side of the hair cells the hair bundle is located. The bundle is formed by stereocilia that are arrayed in rows of increasing height. The stereocilia are connected by means of links. Both the inner and outer hair cells are so-called mechanoelectric transductor (MET) cells. A minimal deflection (1-100 nm) of the stereocilia is transmitted via stereociliary tip links to open the MET channels 3. By opening these MET channels, a positive current of potassium ions flows into the hair cells that subsequently depolarize. Depolarization opens the voltage gated calcium channels, which induces calcium influx. This influx of calcium ions is necessary for the release of glutamate, a neurotransmitter that stimulates the ends of the auditory nerve fibers 4. Figure 2 Organ of corti. This image has been copied by courtesy of J. Brigande and S. Heller 5. 14 | Chapter 1 The deflection of stereocilia of the outer hair cells occurs via direct contact of the hair bundle by the tectorial membrane. The hair bundles of the inner hair cells are thought to be deflected by the fluid movement in the subtectorial space 6. Inner hair cells are responsible for the transduction of sound into a neural signal with high spatial and temporal resolution. Outer hair cells are the cochlear amplifiers and by electromotility enhance the sensitivity, tuning and dynamic range 7. Tonotopy The relation between detection of sounds of specific frequencies and position in the cochlea is called tonotopic organization of the cochlea 8, 9. This tonotopic organization is retained up to the auditory cortex, which enables frequency identification. Due to decreasing stiffness in the basilar membrane, the velocity of the wave decreases, while the amplitude increases, as the sound wave travels through the cochlea. At the point where the amplitude reaches its maximum, the sliding motion between the basilar and tectorial membrane will be the largest. At this position in the cochlea, hair cells will be stimulated. The stiffness of the basilar membrane is the largest at the base (the oval window) and decreases exponentially towards the apex. The hair cells and the associated stereocilia also differ in length from the base to the apex. This difference in height enhances the efficiency of flexoelectricity. The vestibule The vestibular organ is, besides the cochlea, also part of the labyrinth. The vestibular organ consists of three semicircular canals, and two satellite organs: the utricle and saccule. The horizontal, superior and inferior semicircular canals detect angular accelerations and the utricle and saccule detect linear accelerations in the vertical and horizontal plane, respectively 10. Each part of the vestibular organ has its own neuroepithelial area. In the sacculus and utriculus this neuroepithelial areas are called the macula sacculi and macula utriculi, respectively. The neuroepithelial area in each semicircular canal is called crista ampullaris and is located in the ampulla, the dilated end of the semicircular canal. Similar to the cochlea, the neuroepithelium consists of hair cells and supporting cells. At the apex of each hair cell are several stereocilia and one kinocilium located. The stereocilia are arranged evenly in rows of increasing height. Next to the tallest row of stereocilia, the kinocilium is located. The stereocilia are connected by tip-links. When the stereocilia bend, these tip-links open (or close) transduction channels, which results in a change of receptor potential. The stereocilia and kinocilia of the maculae protrude in a gelatinous membrane, which is covered by statoconia (calcium carbonate crystals). Movement of these statoconia causes the stereocilia and kinocilia to bend. The stereocilia and kinocilia in Introduction | 15 the crista ampullaris are also covered by a gelatinous mass which is called the cupula. Angular accelerations of the head cause the cupula to bend, which causes depolarization or hyperpolarization of the hair cells, depending on whether the direction of the deflection is away from or towards the kinocilium. 11. The vestibular organ only detects motion of the head, therefore it is also important to have appropriate information about the orientation of the head in respect to the body. This information comes from proprioceptors in the neck and body. Besides this, visual information is also important in the maintenance of balance 10-12. Figure 3 Human vestibular organ; psc, posterior semicircular canal; hsc, horizontal semicircular canal; asc, ascending semicircular canal; um, utrical macula; pa, posterior ampula. This image has been copied and adjusted by courtesy of Büki et al 13. Hearing impairment Causes of hearing impairment can be divided into conductive and/or sensorineural. A conductive hearing impairment indicates that sound cannot be conducted properly towards the cochlea. In sensorineural hearing impairment the defect is located in the cochlea, the auditory nerve or the further auditory system. Conductive as well as sensorineural hearing impairment can be hereditary or acquired. Of all early onset sensorineural cases of hearing impairment, about half is due to environmental factors and the other half is due to inherited factors 14. The most common cause of acquired early onset hearing impairment is a congenital cytomegalovirus (CMV) infection, which is the causative agent in 10-20% of hearing impaired children. Of all children with a congenital CMV infection, 12.6% will develop hearing impairment 15. Hearing loss may present with a delayed onset, and can be unstable, with fluctuations and progression. Other causes of acquired hearing impairment are trauma, prematurity and ototoxic medication. 16 | Chapter 1 When sensorineural hearing impairment is not acquired, it is inherited. Currently, over 75 genes are known to be expressed in the cochlea and to cause hearing impairment, when mutated 16. Since different genes are known to function in different structures in the cochlea, mutations can cause different types of loss of function of the cochlea. Hereditary hearing impairment When hearing impairment is associated with other distinct features it is called syndromic hearing impairment. These features can change physical appearance, as is for example the case in Treacher Collins, Stickler, Waardenburg or Branchiooto- renal syndrome. These features can also be less obvious or occur at later age and therefore be missed in first respect. Examples of the latter are a prolonged QT-syndrome in Jervell- Lange Nielsen syndrome, retinitis pigmentosa in Usher syndrome or renal failure in Alport syndrome. These syndromes can exhibit all known mendelian, as well as mitochondrial inheritance patterns 17. In the cases of early onset hereditary hearing impairment about 30% is estimated to be syndromic. In these cases, the mutated gene has a significant impact on the function of the ear and another organ or organs 18. Nonsyndromic hearing impairment can present with different inheritance patterns: autosomal recessive (70-80%), autosomal dominant (20-30%), X-linked (<1%), mitochondrial (<1%) or Y-linked (rare) 19, 20. The different genetic loci associated with nonsyndromic sensorineural hearing impairment are defined by DFN (DeaFNess). The inheritance pattern determines the next character; an A for autosomal dominant - DFNA, B for autosomal recessive – DFNB, X for X-linked – DFNX and Y for Y-linked – DFNY. Every locus for hearing impairment is numbered chronologically in order of discovery. Several mutated genes can be causative for syndromic as well as non-syndromic hearing impairment. Good examples are the genes mutated in Usher syndrome type I. MYO7A, CDH23, USH1C, PCDH15, USH1G and CIB2 are all also associated with non-syndromic hearing impairment. Mutations in several specific deafness- associated genes may lead to autosomal dominantly as well as autosomal recessively inherited hearing impairment. This is, amongst others, seen in DFNB7/11 and DFNA36, which are both caused by mutations in TMC1 16. The differences in syndromic/ non-syndromic or autosomal dominant/ recessive inheritance depend on the affected protein domain and the type of mutation 21, 22. In general, mutations can cause: loss of function. gain of function. a dominant negative effect: the product of the mutant gene interferes with the product of the wild type copy of the gene 23. Introduction | 17 Clinical characteristics All but one (MIR9624) of the known deafness- associated genes encode a protein. These proteins may have different functions in the cochlea. The phenotypes associated with different mutated genes may, therefore, also differ. To display these differences correctly, phenotypes are described by means of several characteristics. These features are summarized in table 1. Genetic testing In order to find the genetic defect that underlies hearing impairment one can follow several strategies. Homozygosity mapping In order to identify the region in which the genetic defect is located, one can apply homozygosity mapping. This method is based on the fact that an affected individual, from a consanguineous family, inherits two copies of a disease allele from a common ancestor of father and mother. These copies are therefore homozygous. Since small chromosomal regions intend to inherit as a whole, the nearby marker loci will also inherit in the same way. A marker is a specific DNA sequence with a known location on a chromosome. These homozygous regions will be mapped by means of a single nucleotide polymorphism (SNP) array 26. One can compare the genotypes of two or more family members, and herewith identify regions shared by affected family members. Linkage analysis Linkage analysis is measuring the cosegregation of any DNA sequence variant with a disease. This is also based on the tendency of two or more genetic loci to be transmitted together during meiosis because they are physically close on the chromosome 27. Whether loci are linked or not can be identified by means of polymorphisms, such as single nucleotide polymorphisms (SNPs) 28. It can be used to reject or to prove whether a specific (gene) locus segregates with the disease in a family. Mutation analysis When a gene is suspected to be mutated, one can examine this gene by mutation analysis, e.g. by Sanger sequencing 29. The obtained sequence is compared to the DNA sequence of an unaffected individual 30. Whether a variant is a known common or rare variant can be evaluated in public databases, e.g. the Exome Variant Server 31. The impact of a nucleotide change on gene function can be predicted by different prediction programs, e.g. Mutation Taster 32. 18 | Chapter 1 Table 1 Characteristics to describe hearing impairment Characteristics25 Type of hearing impairment Conductive Sensorineural Mixed Severity (based on the best hearing ear, averaged over 0.5, 1, 2 and 4 kHz) Mild Audiometric configuration Low ascending Moderate Severe Profound Mid frequency (U-shaped) High descending (gently downsloping) High descending (steeply downsloping) Flat Frequency ranges Low Mid High Extended high Symmetry Unilateral - asymmetric Bilateral - symmetric Age of onset Congenital Prelingual Postlingual Progression Variability Interfamilial Intrafamilial Introduction | 19 Associated to disease or deformity of outer/middle ear. Characterized by normal bone-conduction thresholds (<20 dB HL) and an air-bone gap >15 dB HL averaged over 0.5, 1 and 2 kHz. Associated to disease or deformity of the inner ear/auditory nerve Characterized with an air/bone gap < 15 dB HL averaged over 0.5, 1 and 2 kHz. Associated to combined involvement of the outer/middle ear and the inner ear/auditory nerve. Audiometrically >20 dB HL in the bone conduction threshold together with >15 dB HL air-bone gap averaged over 0.5, 1 and 2 kHz. 20-40 dB HL 41-70 dB HL 71-95 dB HL >95 dB HL >15 dB HL difference between the poorer 0.25 and 0.5 kHz thresholds and those at 1, 2, 4 and 8 kHz. >15 dB HL difference between the poorest thresholds of 1 and 2 kHz, and those at 4 and 8 kHz and 0.25 and 0.5 kHz. 15-29 dB HL difference between the mean of 0.5 and 1 kHz and the mean of 4 and 8 kHz. >30 dB HL difference between the mean of 0.5 and 1 kHz and the mean of 4 and 8 kHz. <15 dB HL difference between the mean of 0.25, 0.5 kHz thresholds, the mean of 1 and 2 kHz and the mean of 4 and 8 kHz. ≤ 0.5 kHz >0.5 kHz ≤ 2 kHz >2 kHz ≤ 8 kHz > 8 kHz > 10 dB HL difference between the ears in at least two frequencies. < 10 dB HL difference between the ears in all frequencies. Present at birth Before language development After language development Deterioration of >15 dB HL in the average over the frequencies of 0.5, 1, and 2 kHz within a 10-year period. Differences in above mentioned characteristics between families with hearing impairment based on the same genetic defect. Differences in above mentioned characteristics between family members with hearing impairment based on the same genetic defect. 20 | Chapter 1 Whole exome sequencing Successively sequencing single genes is laborious and time-consuming. Since the beginning of this century novel techniques were introduced that can determine the sequence of all exons (expressed region) by one test: whole exome sequencing (WES) 23. The efficacy of these techniques has been improving gradually. WES has become more cost-effective and result interpretation has been enhanced (i.e. discrimination between a benign variants and causative mutations) over time 33. Novel deafness- associated genes have already been discovered by WES, however, single gene tests are still of practical significance to confirm the causative mutation 34. The aforementioned method of Sanger sequencing seems to become outdated with WES as replacement. However, Sanger sequencing is useful to determine which variant is likely to be causative, and segregates with the disease in the family, or whether a variant is inherited or de novo 27. One can also focus on a specific set of genes via next generation sequencing; this is called targeted next generation sequencing. In 31-60% of the cases of hereditary hearing impairment, the causative mutation is found by targeted next generation sequencing 35. Main goals of otogenetic research There are two main reasons to conduct otogenetic research. The first goal is to be able to counsel a patient and its family on prognosis e.g. progression of the hearing impairment and possible involvement of other organs. The second goal of otogenetic research is to gain insight into the pathophysiological effects of a mutated gene in the cochlea, which might eventually lead to a therapy for specific types of hereditary hearing impairment. Gaining insight in pathophysiological effects is mainly achieved by studying the protein function in the cochlea of mouse and mouse mutants 36. Gaining insight into the function of a protein in the human cochlea is supported by describing a genotype- phenotype correlation, according to the characteristics of table 1. For example, age of onset and progression can provide clues whether a protein is involved in developing a structure or in maintenance of this structure. And by means of vestibular function tests the involvement in the vestibular organ can be determined 37. In order to better categorize hearing impairment and to evaluate cochlear function in detail, de Leenheer et al. introduced the extensive psychophysical test battery from Nijmegen38. Introduction | 21 Psychophysical tests Psychophysical tests evaluate loudness perception, and spectral and temporal processing. Loudness perception is examined by means of loudness scaling. A stimulus is presented with different intensities and the individual is asked to classify these intensities from inaudible to unbearable loud. This test is based on the Würzburger Hörfeld Skalierung developed by Moser 39. Temporal processing is evaluated by means of gap detection. Stimuli are presented randomly with a pause of variable lengths. The test individuals have to report whether a pause is present within the stimulus 40, 41. Spectral processing is estimated by means of a difference limen for frequency test. A stimulus is presented with fluctuation of frequencies. The test person has to indicate whether the fluctuation is present or not 40. These tests are combined with standard pure tone, speech and speech in noise audiometry. Schuknecht and Gacek introduced a categorization of hearing impairment 42. They stated that there are four types of hearing impairment, each correlated to specific structures of the inner ear. These four types are sensory, strial, neural and cochlear conductive. Loss of hair cells would cause a sensory hearing impairment. A strial hearing impairment would be caused by strial atrophy, while loss of cochlear neurons would be the base of a neural hearing impairment. Cochlear conductive hearing impairment is speculated to be caused by alteration in the physical aspects of the cochlear duct 42. Two of these four types have been identified by psychophysical testing. Patients with Usher syndrome type 2a have been subjected to these psychophysical tests. When compared to individuals with sensory hearing loss, comparable results were observed. A sensory type of hearing impairment would be expected since USH2A is part of the Usher interactome, which is essential for the morphogenesis of the hair bundle 43. In patients with mutations in TECTA (DFNA8/12), a cochlear conductive hearing impairment has been demonstrated. A parallel shift of the curves during loudness scaling was seen, combined with normal ranged results of gap detection, difference limen for frequency and speech reception in noise. DFNA8/12 patients perform comparable to patients with DFNA13 (caused by mutations in COL11A2) 38, 44. Both TECTA and COL11A2 are expressed in the tectorial membrane. Impaired protein function can cause tectorial membrane dysfunction 45. These results are also seen in patients with a conductive hearing loss where processing is not affected but transduction of sound is. These examples demonstrate the pathological effect of a mutated gene on the specific structures in the cochlea. This knowledge can be important in the application of different types of rehabilitation. 22 | Chapter 1 Counseling When the genetic defect has been identified, the patient needs adequate counseling. Genetic counseling is needed in order to communicate information about the nature of the disease, inheritance mode and implications for possible mutation carriers in the family 46. The nature of the disease includes the degree of hearing impairment, prognosis and expectations on rehabilitation options 47, but also possible additional symptoms. In this process, one should take cultural aspects, socials needs and educational level into account 46. The nature of the disease is important because it not only influences rehabilitation but also occupational choices. In patients with syndromic hearing impairment counseling on early detection and possible treatment of additional symptoms is necessary. In addition, prevention or slowing down the progression of hearing loss might be possible, for example in patients with an enlarged vestibular aqueduct or with an elevated susceptibility for aminoglycoside induced hearing impairment 48. Not much research has been done on the, mainly psychosocial, consequences of genetic testing for hereditary hearing impairment 49. A recent evaluation, however, suggests an increase in psychosocial well being after counseling on a positive genetic test 50. When hearing loss has been diagnosed and examined, rehabilitation options have to be considered. Rehabilitation options for hereditary hearing impairment in general vary from FM (frequency modulation) equipment for mild types of impairment, hearing aids for mild to severe hearing losses and cochlear implantation for severe to profound types of hearing impairment. Different studies have focused on the results of rehabilitation in hereditary hearing impairment. These studies are mainly evaluating cochlear implantation. The results are difficult to interpret because the postoperative performance in patients after cochlear implantation is influenced by many factors. DFNB1 is, because of its high prevalence, the most commonly studied type of hereditary hearing impairment with regard to results of cochlear implantation. Compared to non-DFNB1 patients, patients with DFNB1 present with similar or better performance results after cochlear implantation 51. Eppsteiner et al. state that genetic defects that affect the spiral ganglion may result in worse performance after cochlear implantation. This is supported by results of two patients with mutations in TMPRSS3 52. However, Weegerink et al. reported seven patients with fairly good speech recognition scores one year after implantation 53. When such good results are achieved, Eppsteiner et al. hypothesize that the initial good performance will diminish over time 52. The conclusion of this spiral ganglion hypothesis can only be confirmed when larger numbers of patients are available, including an extensive follow up. Introduction | 23 In general, the first results of cochlear implantation appear to be good in patients with hereditary hearing impairment. However, more research needs to be done to second this 54. The results of these studies are, amongst others, important for counseling a patient on its expectations on performance after cochlear implantation. Additionally, the right type and early start of rehabilitation is important for an adequate speech and language development, but in general also for cognitive and psycho-affective development 55. Research on the different rehabilitation options in hereditary hearing impairment therefore deserves more attention. Aim of this thesis This thesis is aimed to contribute to the expanding knowledge on effects of mutated deafness genes on hearing. As a result, this expansion of knowledge will improve counseling on hereditary hearing impairment. The research described in this thesis is focused on providing an overview of several types of currently known autosomal recessive hereditary nonsyndromic types of hearing impairment. In addition, novel genotype-phenotype correlations of autosomal recessive hearing impairment types are described. This thesis also contributes to expanding an already known genotype-phenotype correlation, that of DFNB1. Since the knowledge on hereditary hearing impairment is expanding, the psychosocial impact of an otogenetic diagnosis was evaluated as well. Thesis outline 1.2 An overview of all, currently known autosomal recessive types of hereditary hearing impairment. 2.1 OTOG and OTOGL are two deafness- associated genes that show large similarities in expression and structures of the encoded protein. The audiometric characteristics of several families with mutations in either of these two genes are evaluated and compared. In addition, psychophysical tests were performed to evaluate the effect of mutations in these genes on the function of the cochlea. 2.2 A novel deafness- associated gene, CLIC5, which is expressed in several organs, is found to be associated with autosomal recessive hearing impairment. In this chapter, phenotypic and genotypic features of mutations in CLIC5 are delineated. 2.3 USH1G is one of the genes known to cause Usher syndrome type I, when mutated. All other genes involved in Usher syndrome type 1 are also known to be involved in nonsyndromic hearing impairment. The audiometric, vestibular 24 | Chapter 1 3. 4. 5. and ophthalmologic characteristics of an autosomal recessive nonsyndromic form of hearing impairment caused by mutations in USH1G are described. DFNB1 is the most common type of autosomal recessively inherited hearing impairment. This type of hearing impairment is, therefore, extensively studied. Inconsistent results on vestibular function and imaging characteristics in DFNB1 are described in literature. In this chapter, vestibular function in combination with temporal bone imaging in DFNB1 patients are evaluated. Knowledge on different types of hereditary hearing impairment is expanding, however, knowledge on the impact of an otogenetic diagnosis lags behind. This chapter focuses on the psychosocial impact of an otogenetic diagnosis on patients with hearing impairment. General discussion Introduction | 25 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Taselaar M. Over binaurale selectieve versterking en de verstaanbaarheid van spraak 1959 [[31-12-2014]. Available from: http://www.audiologieboek.nl. Guyton AC, Hall JE. The Sense of Hearing. Textbook of medical physiology. 112006. p. 651-62. Fettiplace R, Kim KX. 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Audiology & neuro-otology. 2011;16(2):93-105. Riazuddin S, Nazli S, Ahmed ZM, et al. Mutation spectrum of MYO7A and evaluation of a novel nonsyndromic deafness DFNB2 allele with residual function. Human mutation. 2008;29(4):502-11. Strachan T, Read A. Human Molecular Genetics. New York: Garland Science; 2011. Solda G, Robusto M, Primignani P, et al. A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing. Human molecular genetics. 2012;21(3):577-85. 26 | Chapter 1 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. Mazzoli M, van Camp G, Newton V, et al. Recommendations for the description of genetic and audiological data for families with nonsyndromic hereditary hearing impairment. available at http:// hereditaryhearinglossorg/. Accessed december 2014. Génin E, Todorov AA. Homozygosity Mapping. eLS: John Wiley & Sons, Ltd; 2001. Bailey-Wilson JE, Wilson AF. Linkage analysis in the next-generation sequencing era. Human heredity. 2011;72(4):228-36. Mueller RF, Young ID. Emery’s Elements of Medical Genetics: Harcourt Publishers Limited; 2001. Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. Journal of molecular biology. 1975;94(3):441-8. Jorde LB, Carey JC, Bamshad MJ, et al. Medical Genetics: Mosby; 2003. (ESP) NESP. Exome Variant Server 2015. Available from: http://evs.gs.washington.edu/EVS/. Schuelke M, Schwarz JM, Seelow D. Mutation Taster. Available from: http://www.mutationtaster.org. Neveling K, Feenstra I, Gilissen C, et al. A Post-Hoc Comparison of the Utility of Sanger Sequencing and Exome Sequencing for the Diagnosis of Heterogeneous Diseases. Human mutation. 2013;34(12): 1721-6. Diaz-Horta O, Duman D, Foster J, 2nd, et al. Whole-exome sequencing efficiently detects rare mutations in autosomal recessive nonsyndromic hearing loss. PloS one. 2012;7(11):e50628. Atik T, Bademci G, Diaz-Horta O, et al. Whole-exome sequencing and its impact in hereditary hearing loss. Genetics research. 2015;97:e4. Friedman LM, Dror AA, Avraham KB. Mouse models to study inner ear development and hereditary hearing loss. The International journal of developmental biology. 2007;51(6-7):609-31. Schraders M, Oostrik J, Huygen PL, et al. Mutations in PTPRQ are a cause of autosomal-recessive nonsyndromic hearing impairment DFNB84 and associated with vestibular dysfunction. American journal of human genetics. 2010;86(4):604-10. De Leenheer EM, Bosman AJ, Kunst HP, et al. Audiological characteristics of some affected members of a Dutch DFNA13/COL11A2 family. The Annals of otology, rhinology, and laryngology. 2004;113(11): 922-9. Moser MM. Das Würzburger Hörfeld, ein Test für prothetische Audiometrie. HNO. 1987;35:318-21. Oonk AM, Leijendeckers JM, Huygen PL, et al. Similar phenotypes caused by mutations in OTOG and OTOGL. Ear and hearing. 2014;35(3):e84-91. Moore BC. Temporal analysis in normal and impaired hearing. Annals of the New York Academy of Sciences. 1993;682:119-36. Schuknecht HF, Gacek MR. Cochlear pathology in presbycusis. The Annals of otology, rhinology, and laryngology. 1993;102(1 Pt 2):1-16. Leijendeckers JM, Pennings RJ, Snik AF, et al. Audiometric characteristics of USH2a patients. Audiology & neuro-otology. 2009;14(4):223-31. Plantinga RF, Cremers CW, Huygen PL, et al. Audiological evaluation of affected members from a Dutch DFNA8/12 (TECTA) family. Journal of the Association for Research in Otolaryngology: JARO. 2007;8(1):1-7. Legan PK, Lukashkina VA, Goodyear RJ, et al. A deafness mutation isolates a second role for the tectorial membrane in hearing. Nature neuroscience. 2005;8(8):1035-42. ACMG. Genetics Evaluation Guidelines for the Etiologic Diagnosis of Congenital Hearing Loss. Genetic Evaluation of Congenital Hearing Loss Expert Panel. ACMG statement. Genetics in medicine : official journal of the American College of Medical Genetics. 2002;4(3):162-71. Wu CC, Liu TC, Wang SH, et al. Genetic characteristics in children with cochlear implants and the corresponding auditory performance. The Laryngoscope. 2011;121(6):1287-93. Matsunaga T. Value of genetic testing in the otological approach for sensorineural hearing loss. The Keio journal of medicine. 2009;58(4):216-22. Broadstock M, Michie S, Marteau T. Psychological consequences of predictive genetic testing: a systematic review. European journal of human genetics : EJHG. 2000;8(10):731-8. Palmer CG, Boudreault P, Baldwin EE, et al. Deaf genetic testing and psychological well-being in deaf adults. Journal of genetic counseling. 2013;22(4):492-507. Introduction 51. 52. 53. 54. 55. | 27 Varga L, Kabatova Z, Masindova I, et al. Is deafness etiology important for prediction of functional outcomes in pediatric cochlear implantation? Acta oto-laryngologica. 2014;134(6):571-8. Eppsteiner RW, Shearer AE, Hildebrand MS, et al. Prediction of cochlear implant performance by genetic mutation: the spiral ganglion hypothesis. Hearing research. 2012;292(1-2):51-8. Weegerink NJ, Schraders M, Oostrik J, et al. Genotype-phenotype correlation in DFNB8/10 families with TMPRSS3 mutations. Journal of the Association for Research in Otolaryngology: JARO. 2011; 12(6):753-66. Vivero RJ, Fan K, Angeli S, et al. Cochlear implantation in common forms of genetic deafness. International journal of pediatric otorhinolaryngology. 2010;74(10):1107-12. Tomblin JB, Oleson JJ, Ambrose SE, et al. The influence of hearing aids on the speech and language development of children with hearing loss. JAMA otolaryngology-- head & neck surgery. 2014;140(5): 403-9. 1.2 Features of autosomal recessive nonsyndromic hearing impairment; a review to serve as a reference A.M.M. Oonk P.L.M. Huygen H.P.M. Kunst H. Kremer R.J.E. Pennings Clinical Otolaryngology, Epub ahead of print 30 | Chapter 1 Abstract Nonsyndromic sensorineural hearing impairment is inherited in an autosomal recessive fashion in 75-85% of cases. To date, 61 genes with this type of inheritance have been identified as related to hearing impairment, and the genetic heterogeneity is accompanied by a large variety of clinical characteristics. Adequate counseling on a patient’s hearing prognosis and rehabilitation is part of the diagnosis on the genetic cause of hearing impairment and, in addition, is important for the psychological well-being of the patient. All articles describing clinical characteristics of the audiovestibular phenotypes of identified genes and related loci have been reviewed. This review aims to serve as a summary and a reference for counseling purposes when a causative gene has been identified in a patient with a nonsyndromic autosomal recessively inherited sensorineural hearing impairment. Introduction | 31 Prevalence and causes of hearing impairment Hearing impairment is the most prevalent sensorineural disorder in developed countries and also the most common birth defect. Approximately one out of every 1,000 newborns is born profoundly deaf and one out of every 300 newborns has a permanent mild-to-profound congenital hearing impairment. In addition, for every ten congenitally hearing impaired children, five to nine children will develop a similar hearing loss before adulthood1-3. Globally, 360 million people have a hearing impairment, which is defined as a hearing loss greater than 40 dB HL 4. Hearing impairment can be acquired or inherited. In about half of early-onset cases, it is inherited. Noise exposure is the most common environmental factor that contributes to acquired hearing loss in adults. An individual’s susceptibility to hearing loss caused by noise exposure is, in addition, a good example of gene-environment interaction 1. Hereditary hearing impairment More than half of prelingual hearing impairment cases occur due to genetic factors. In 30% of cases, the hearing impairment is syndromic and accompanied by other symptoms, and in 70% of cases it is nonsyndromic. The latter group of hearing impairments can be divided by the mode of inheritance. Nonsyndromic hearing impairments are autosomal recessively inherited in 75-85% of cases and autosomal dominantly inherited in 15-24% of cases. The remaining 1-2% demonstrate mitochondrial or X- or Y-linked inheritance. Currently, over 80 genes and over 140 loci have been identified for nonsyndromic hearing impairment 5. The loci are defined by the mode of inheritance and are designated by the letters DFN (DeaFNess). DFN is followed by an A for autosomal dominant inheritance (DFNA) and a B for autosomal recessive inheritance (DFNB). Both loci are affixed with a number based on their order of identification/publication. Hereditary hearing impairments with an X-linked inheritance are designated DFNX 2, 5-7. So far, 61 genes have been identified for autosomal recessively inherited nonsyndromic hearing impairment (arNSHI) 5. arNSHI is therefore highly heterogeneous, making it difficult to give an overview of all of the related phenotypes. 32 | Chapter 1 Obtaining a genetic diagnosis of hereditary hearing impairment A genetic diagnosis of hereditary hearing impairment is currently much easier established in the out-patient setting than it was a decade ago. An increasing number of genes can now be sequenced on a regular basis, and high-throughput techniques, such as massive parallel sequencing (e.g. Affymetrix array), are being introduced in the clinic and will lead to easier identification of causative genes in the near future. This is especially important for the identification of autosomal recessive sensorineural hearing impairment in isolated cases and is therefore an important step forward in obtaining a genetic diagnosis on hearing impairment in these cases. In the Western world, most families are small and most cases, therefore, are isolated. In these cases, other causes appear to be more obvious and it is difficult to identify underlying genetic defects. Discovery of the genetic causes of hearing impairment and subsequent counseling of patients has been shown to improve scores on the scale of perceived personal control, depression and anxiety. In other words, these patients experience an increase in their degree of psychological well-being 8. It is important to know the genetic diagnosis if one wants to counsel patients on, for example, prognosis of hearing impairment (“will my hearing loss progress or not?”) on the outcomes of rehabilitation with, for example, cochlear implantation and on the consequences for potential progeny 9. Purpose of this review This review presents a summary of the clinical characteristics of all currently known nonsyndromic autosomal recessively inherited types of sensorineural hearing impairment. For this purpose, all articles describing clinical characteristics of the audiovestibular phenotype of identified genes and related loci have been reviewed. This paper can be used for counseling purposes with patients in whom the causative gene for autosomal recessive sensorineural hearing impairment has been identified. The focus of this review is only on this type of hearing impairment. Other types, such as dominant types, syndromic types and mixed hearing impairment are outside its scope. However, it should be kept in mind that syndromic hearing impairment can present at first with hearing impairment as its only feature. Each type of arNSHI will be described by its age of onset, severity, progression, audiogram configuration and associated vestibular function. The severity, affected frequencies and audiogram configurations will be described according to GENDEAF recommendations, which are described in Tables 1, 2 and 3, respectively 10. Introduction Table 1 Severity of hearing impairment | 33 Table 2 Frequency ranges Severity Pure Tone Average Frequencies Mild 20-40 dB HL Low ≤ 0.5 kHz Moderate 41-70 dB HL Mid >0.5 ≤ 2 kHz Severe 71-95 dB HL High >2 ≤ 8 kHz Profound >95 dB HL Extended high >8 kHz Severity of hearing impairment (table 1) and frequency ranges (table 2) according to GENDEAF recommendations 9. Table 3 Audiogram configurations Configuration Low frequency ascending >15 dB HL difference between the (poorer) lower frequencies to the higher frequencies Mid frequency U-shaped (or cookie bite) >15 dB HL difference between the (poorer) mid frequencies and the lower and higher frequencies Gently downsloping 15-29 db HL difference between the mean of 0.5 and 1 kHz and the (poorer) mean of 4 and 8 kHz Steeply downsloping >30 dB HL difference between the mean of 0.5 and 1 kHz and the (poorer) mean of 4 and 8 kHz Flat <15 dB HL difference between the mean of 0.25 and 0.5 kHz, the mean of 1 and 2 kHz and the mean of 4 and 8 kHz. Definition of different audiogram configurations according to GENDEAF recommendations 9. First, the most common type of arNSHI, DFNB1, will be described. This will be followed by a description of the most prevalent phenotype of arNSHI: prelingual profound hearing impairment. The remaining phenotypes, with different, more specific characteristics, will then be described and, finally, two auditory neuropathy spectrum disorders with autosomal recessive inheritance will be described. GJB2/GJB6 (DFNB1) DFNB1 is caused by mutations in GJB2 and/or GJB6. Both genes are found at the same locus, and mutations in both genes can cause hearing impairment. It was suggested that DFNB1 may have a digenic inheritance pattern; however, the data of Wilch et al. indicate that it does not 11. DFNB1 is known for its high prevalence around the world. The percentage of hereditary hearing impairments caused by mutations in GJB2/GJB6 varies by geographical location. A range of 18-41% has been reported 34 | Chapter 1 for Europe, Northern America, Australia, the Middle East and East Asia. In Southern Europe, GJB2 mutations are found more frequently than in Northern or Central Europe (where carrier frequencies are 1:35 and 1:79, respectively). The latter correlates with the prevalences found in North America and Australia 12. DFNB1 is encountered less frequently in Iran and India (15%), and even less so in Pakistan (6%) 13,14. The high carrier prevalence has led different studies to recommend sequencing of GJB2 and screening for deletions in GJB6 as a first step in cases of suspected autosomal recessive inheritance or in isolated cases of congenital or prelingual nonsyndromic hearing impairment 15, 16. This strategy has a sensitivity of over 98%. Therefore, when no mutations are found with this strategy, other genetic causes are highly likely 17. DFNB1 has most often a congenital and prelingual onset. In most cases, hearing impairment in DFNB1 is congenital and is reported to be stable or only slightly progressive. However, when the onset is not congenital, hearing impairment can be very progressive. The audiogram configuration is flat to downsloping, though this is not pathognomonic. There is a large variability in severity that is reported to range from mild to profound 14. Snoeckx et al. 18 concluded that truncating mutations are associated with a higher degree of hearing impairment than non-truncating mutations in a large multicenter study of 1,531 DFNB1 patients. In addition, the authors also stated that the pathogenicity of missense mutations depends on the position and nature of the substitution. This is one of the possible explanations for the substantial phenotypic variability among cases with different mutations in the same gene, which is not only applicable to DFNB1. Due to the large variability in phenotypes, it may be difficult to give prognostic information regarding hearing impairments 19. Such information should only be provided to the DFNB1 patient who carries a specific combination of GJB2/GJB6 mutations for which reliable evidence regarding the genotype-phenotype correlation is available. The most prevalent and important mutation combinations have been explored in this respect in the multicenter study by Snoeckx et al. 18. The predominant phenotype was associated with homozygous 35delG mutations: the audiometric configuration was fairly similar to the one shown in Fig. 2D for CDH23 mutations. So far, only limited research on vestibular function in DFNB1 patients has been published, which is remarkable considering its high prevalence. There is no consensus in the literature on vestibular function in DFNB1 20-22. Further evaluation of vestibular function in larger numbers of DFNB1 patients is clearly needed to reach a more definitive conclusion on vestibular function in DFNB1. Introduction | 35 Severe to profound hearing impairment Aside from GJB2/GJB6 (DFNB1), 31 other genes have been identified that can be associated with prelingual severe to profound sensorineural hearing impairments. Supplemental Table 1 describes the phenotypes of these genes when they have mutations. Because all of these phenotypes are described as severe to profound hearing impairments, it is difficult to clinically differentiate between them. In addition, the manner in which the severity of each hearing impairment was assessed is often not clearly described, and audiograms are also frequently missing in articles reporting on the identification of novel deafness– associated genes. Some of these genes will be discussed briefly here due to their relatively high prevalence of mutations, reported variable onset of the hearing impairment and associated vestibular dysfunction. MYO15A (DFNB3) MYO15A (DFNB3) is, after GJB2/GJB6, the gene that has been found to be mutated most often in patients with prelingual, severe to profound sensorineural arNSHI without vestibular dysfunction. So far, approximately 28 different mutations of this gene have been reported in more than 50 families 6. TMC1 (DFNB7/11) Another frequent cause of prelingual or postlingual severe to profound sensorineural arNSHI are mutations in TMC1 (DFNB7/11)23. Thus far, approximately 21 different mutations have been reported and over 50 families with prelingual severe to profound hearing impairments due to DFNB7/11 have been described. This is similar to the phenotype seen in TMC1 knockout mice 24. DFNB7/11 can also present with postlingual hearing impairment, as described by de Heer et al. (Supplemental table 1). The hearing impairment in this specific family occurs after 4 years of age in the high frequencies and progresses rapidly in the first 3 decades to a profound hearing impairment. The authors suggest that this may be related to mutations that do not completely abolish normal splicing of TMC1 mRNA or to a truncated protein that still has some residual function 25. MYO7A (DFNB2) It has been reported that severe-to-profound sensorineural hearing impairment due to DFNB2 can have a congenital onset but can also go unnoticed until up to 16 years of age. Balance problems and vestibular dysfunction on caloric testing have been described 26. DFNB2 has only been described in seven families and not in the Caucasian population 27. In addition to DFNB2, mutations in MYO7A can also cause Usher syndrome type 1b, a congenital form of deafblindness with profound deafness, 36 | Chapter 1 vestibular areflexia and retinitis pigmentosa as characteristic features. Riazuddin et al. stated that DFNB2 is associated with residual protein function of myosin VIIA, whereas mutations in MYO7A that cause Usher syndrome type 1b lead to no residual function of the mutated protein 28. ESPN (DFNB36) and MYO6 (DFNB37) Mutations in ESPN and MYO6 result in a phenotype with severe to profound prelingual sensorineural hearing loss and variable vestibular function, ranging from normal to complete vestibular areflexia. In addition to this, mutations in MYO6 are also reported to cause mild, not further specified, facial dysmorphic features. Defects in MYO6 have only been reported to cause arNSHI in 3 Pakistani families. DFNB36 has only been described in three families from Pakistan and Morocco 29-31. Autosomal recessive hearing impairment with characteristic features (Figure 1A-D). STRC (DFNB16) Patients with hearing impairment caused by mutations in STRC generally have a mild to moderate, gently downsloping audiogram. The hearing impairment has an early childhood onset and remains stable over time. It can, however, also be more severe (up to profound). Vestibular abnormalities are not reported 32. Francey et al. suggest that STRC mutations might greatly contribute to mild to moderate hearing impairments in the pediatric population worldwide, because the prevalence of STRC deletions in a GJB2 negative cohort was approximately 5.5% 33. Thus far, 20 families with DFNB16 have been identified. GRXCR2 (DFNB101) DFNB101 is another recently discovered type of arNSHI that presents with moderate hearing loss and a gently downsloping audiogram configuration. The hearing impairment is usually diagnosed at the age of two years and is reported as being progressive. Balance problems have not been observed 34. OTOGL (DFNB84B) Mutations in OTOGL cause a prelingual hearing impairment; one patient failed the neonatal hearing screening and his siblings were subsequently diagnosed with hearing loss at 2 and 3 years of age. The hearing impairment is mild to moderate and stable, and has a flat to gently downsloping configuration. Vestibular hypofunction is seen in DFNB84B patients, resulting in delayed motor milestones 35. DFNB84B is phenotypically similar to DFNB18B. Introduction | 37 OTOG (DFNB18B) DFNB18B presents with a prelingual sensorineural hearing impairment that causes delayed speech development. The hearing impairment begins as mild to moderate but can be slightly progressive after the second decade 35. The audiogram configuration is flat to gently downsloping. Delayed motor milestones have been reported and bilateral weakness has been observed during caloric testing. The resemblance between DFNB18B and DFNB84B is probably a result of similarities in the structure and expression of otogelin and otogelin-like, the protein products of OTOG and OTOGL, respectively, in the inner ear. GJB3 GJB3 is reported to be primarily seen in non-consanguineous Chinese families. The related hearing impairment occurs prelingually or in early childhood and is moderate in severity with a flat audiogram configuration. Vestibular problems have not been reported 36. A dB -10 0 CABP2 CDH23 GIPC3 GJB3 GRXCR1 OTOG OTOGL PJVK PTPRQ SERPINB6 TECTA 20 40 60 80 100 120 140 .25 20 CABP2 GIPC3 TECTA 40 60 80 100 120 140 .5 1 2 4 CDH23 CLIC5 GIPC3 GRXCR1 GRXCR2 LOXHD1 MYO3A OTOG OTOGL PJVK SERPINB6 SLC26A4 STRC USH1G 20 40 60 80 100 120 140 .5 1 2 4 .25 8 kHz C dB -10 0 .25 B dB -10 0 8 kHz .5 1 2 4 8 kHz D dB -10 0 20 40 60 80 100 CLIC5 SERPINB6 TMPRSS3 120 140 .25 .5 1 2 4 8 kHz Figure 1 A-D Examples of audiogram configurations. The Y-axis indicates hearing thresholds (dB HL). On the right, the genes that, when mutated, can present with that type of audiogram configuration are listed. The X-axis indicates thefrequencies (kHz). A. Flat. B. U-shape. C. Gently downsloping. D. Steeply downsloping in the high frequencies. 38 | Chapter 1 GRXCR1 (DFNB25) In DFNB25, patients have a moderate to severe hearing impairment with a flat to gently downsloping audiogram configuration. This can progress until the hearing impairment is profound. This hearing impairment is defined as congenital because the patients failed their neonatal hearing screenings or had delayed speech development. The hearing thresholds of patients with DFNB25 are displayed in figure 2A. Vestibular dysfunction is noted in some DFNB25 patients, who had normal motor milestones with later difficulties in motor development or abnormal rotatory chair test results 37. USH1G A downsloping audiogram configuration is also seen in patients with hearing impairment due to mutations in USH1G. One Dutch family with two affected siblings presented with a nonsyndromic, moderate hearing impairment with an onset at around 4 years of age. A mild progression in hearing thresholds, especially in the higher frequencies, was found. Electronystagmography showed normal vestibular responses to rotatory and caloric stimulation. An ophthalmologic examination found no abnormalities, and, specifically, no signs of retinitis pigmentosa 38. LOXHD1 (DFNB77) When LOXHD1 is the causative gene for hearing impairment, the audiogram shows a flat configuration in all frequencies or a downsloping configuration in the lower frequencies and flat configuration in the mid and high frequencies. The hearing impairment is reported to be congenital as well as postlingual (at 7-8 years of age), and its level is moderate but can progress to a profound hearing impairment. Age Related Typical Audiograms (ARTA) for DFNB77 are shown in figure 2B. DFNB77 has, so far, only been reported in 6 families, including two Ashkenazi Jewish families 39-42. TECTA (DFNB21) When TECTA is mutated, a prelingual moderate to severe hearing impairment is reported. DFNB21 presents with a distinct U-shaped audiogram configuration and is stable 43. It has been reported to present unilaterally in one DFNB21 patient 44. CAPB2 (DFNB93) A flat to U-shaped audiogram can also be seen in patients with DFNB93. The level of the hearing impairment is moderate to severe, and it is stable and reported to have a prelingual onset 45, 46. Introduction A dB -10 0 B dB -10 0 GRXCR1 C dB -10 0 LOXHD1 ATD 1.1-1.7 (n.s.) 20 20 20 40 40 40 60 60 80 80 100 100 120 120 140 140 .25 .5 1 2 4 8 kHz 120 .5 1 2 4 8 kHz .25 20 40 40 40 60 60 60 80 80 80 100 100 120 120 140 140 2 4 .25 8 kHz 4 .5 1 8 kHz MYO3A ATD 0.9-1.2 S 0.5, 8 kHz 30 40 50 60 70 2 4 8 kHz 120 140 .25 .5 1 2 4 8 kHz H dB -10 0 SERPINB6 ATD 0.1-1.7 (n.s.) 20 2 100 10 20 30 G dB -10 0 1 F dB -10 0 20 1 .5 E SLC26A4 20 .5 10 20 30 40 50 60 80 100 140 .25 dB -10 0 CDH23 .25 PTPRQ ATD 0.9-1.3 (all S) 60 20 30 40 50 60 D dB -10 0 | 39 40 60 60 25 35 45 55 100 120 ATD 0.1-1.6 S 2-4 kHz 20 40 80 PJVK 140 5 15 25 80 100 120 140 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz Figure 2 A. Mean thresholds and standard deviations of all known patients with DFNB25 (GRXCR1), age ranges from 10-25 years. B. Age Related Typical Audiograms (ARTA) for DFNB77 (LOXHD1). C. ARTA for DFNB84A (PTPRQ). Significant Annual Threshold Deterioration (ATD, in dB per year) is shown in the graph. D. Mean thresholds and standard deviations for all known patients with DFNB12 (CDH23). E. ARTA for DFNB4 (SLC26A4). F. ARTA for DFNB30 (MYO3A), significant ATD at 0.5 and 8 kHz. G. ARTA for DFNB91 (SERPINB6); ATD is not significant. H. ARTA for DFNB59 (PJVK) with significant ATD at 2 and 4 kHz. 40 | Chapter 1 GIPC3 (DFNB15/72/95) Mutations in GIPC3 cause an early onset (3-11 months of age) hearing impairment that can present with several audiogram configurations, including flat, gently downsloping and U-shaped. The hearing impairment is reported to be stable. The level of the hearing impairment is generally severe to profound, but moderate levels have also been observed. Vestibular problems have not been reported 47. TMPRSS3 (DFNB8/10) Mutations in TMPRSS3 can present with a very characteristic steeply sloping or ski-slope audiogram configuration (figure 1D). In our clinic, approximately 25% of arNSHI that display a ski slope audiogram configuration can be explained by mutations in TMPRSS3 [unpublished data]. The hearing impairment is progressive and eventually leads to a flat profound hearing loss. It is designated as DFNB8 when the onset of the hearing impairment is postlingual (5-12 years) and DFNB10 when the onset is prelingual (0-1.5 years). The severity of mutations in TMPRSS3 predicts how the hearing impairment will present. When mutations cause a greater change within the protein, patients can present with a prelingual severe to profound hearing impairment without the characteristic audiogram configuration. Balance problems have not been reported. Although Weegerink et al. observed vestibular hyporeflexia and hyperreflexia during electronystagmography in some cases, the overall prevalence was not above chance level 48. PTPRQ (DFNB84A) DFNB84A is characterized by a congenital, moderate hearing impairment with a flat configuration that deteriorates over time to a profound hearing impairment (figure 2C). Normal motor milestone are reported; however, vestibular dysfunction (hyporeflexia or areflexia) may occur. Another feature is that variation is seen in the severity of hearing impairments and vestibular problems between affected family members within one family 49, 50. CLIC5 (DFNB102) Mutations in CLIC5 present with an early onset hearing impairment; patients passed the neonatal hearing screening but failed otoacoustic emissions (OAEs) testing after 4 months. The hearing impairment is initially mild and progresses within the first decade to severe to profound levels. The audiogram configuration is gently to steeply downsloping. The vestibular phenotype is probably also progressive because motor milestones were not delayed, however later in life balance problems occurred. Electronystagmography showed complete vestibular areflexia and associated balance problems with increasing age. In addition, signs of a mild subclinical nephropathy were seen in one patient 51. Introduction | 41 SYN4 (DFNB76) DFNB76 presents with an early onset (congenital- 6 years of age), progressive hearing impairment. The audiogram is configuration is steeply sloping. DFNB76 has been reported in two Iraqi Jew families52. CDH23 (DFNB12) Another type of arNSHI that may present with a progressive hearing impairment, though without vestibular problems, is DFNB12. DFNB12 is a prelingual hearing impairment that mainly affects the high frequencies, but the audiogram configuration can range from flat to downsloping. The hearing impairment may progress from moderate to profound (figure 2D); however, the hearing impairment can also be stable and severe to profound 53. Miyagawa et al. reported that the onset of the hearing impairment can occur at a later age (2-60 years of age) in addition to a prelingual onset 54. Vestibular problems were not reported 55. Because CDH23 is also involved in Usher syndrome type 1D, an ophthalmologic evaluation should be considered. Pennings et al. reported abnormal fundoscopic findings in two DFNB12 patients who were not typical for retinitis pigmentosa, and the patients had normal vision 53. Mutations in CDH23 have been described frequently in arNSHI patients and in the Caucasian population 6. SLC26A4 (DFNB4) Worldwide, DFNB4 is, like DFNB1, a common cause of deafness 6. It is usually characterized by an enlarged vestibular aqueduct (EVA) or an incomplete partition type II (classic Mondini dysplasia). These temporal bone abnormalities may occur unilaterally or bilaterally 56, 57 and cause progressive, fluctuating or stable hearing impairments that may present at birth. Hearing impairments range from mild to profound and may be purely sensorineural or mixed in nature (figure 2E). The latter occurs in combination with a normal tympanogram 58. Head trauma, even if mild, can lead to a sudden loss of hearing 59. Vestibular symptoms, such as vertigo, may also occur. Mutations in SLC26A4 can also cause Pendred syndrome, which is marked by the presence of thyroid dysfunction and/or possible development of goiter. Differentiating between DFNB4 and Pendred syndrome can be difficult at young ages because the thyroid dysfunction usually appears in the second decade 58, 60, 61. MYO3A (DFNB30) One consanguineous Iraqi family has been reported to have DFNB30. The hearing impairment often presented in the second decade, but the age of onset varied between family members. A moderate hearing impairment that progressed to a profound hearing impairment, with a downsloping audiogram configuration, was observed (figure 2F). No vestibular problems were reported 62. 42 | Chapter 1 BDP1 (DFNB49) A consanguineous family from Qatar was described to have a post-lingual, moderate to severe, mildly progressive hearing impairment. The audiogram demonstrated a gently downsloping configuration 63. SERPINB6 (DFNB91) DFNB91 is reported to have an onset at over 20 years of age. The hearing impairment is moderate to severe, and flat or gently to steeply downsloping audiogram configurations have been observed. This hearing impairment is progressive (figure 2G) and vestibular symptoms have not been reported. Thus far, this type has only been found in one consanguineous Turkish family 64. Autosomal recessive auditory neuropathy spectrum disorder An auditory neuropathy is characterized by absent auditory brainstem responses and intact OAEs and/ or cochlear microphonics 65. These OAEs may disappear at later age 66. One should be aware that children with an auditory neuropathy spectrum disorder (ANSD) may be missed at neonatal hearing screenings because, in many countries, screening is performed using OAEs 66, 67. Currently, mutations in two genes are known to cause ANSD with prelingual mild to profound hearing impairments (Supplemental Table 3). DFNB9 (OTOF) occurs as a prelingual (<2 years) mild to profound hearing impairment 66, 68. Mutations in OTOF are frequently found in autosomal recessive hearing impairments 6. Although one would be inclined to expect unfavorable results with cochlear implantation in patients with ANSD, good post-implant results have been reported for patients with DFNB9 69, 70. Mutations in OTOF can also cause temperature-sensitive nonsyndromic ANSD. Patients with this type of ANSD have normal hearing or a mild hearing impairment when afebrile. When febrile, hearing levels are reduced to a severe or profound level and return to normal when the fever diminishes 71. Another gene that is involved in ANSD is PJVK (DFNB59). DFNB59 is known for causing a prelingual moderate to profound sensorineural hearing impairment. The hearing impairment can be stable or progress over time. The audiogram configuration is flat to gently downsloping (figure 2H). DFNB59 does not always present as ANSD. Delmaghani et al. have, in fact, been the only researchers to report that OAEs were present 72. Vestibular dysfunction is not commonly noted in patients with PJVK mutations, but central vestibular dysfunction was reported by Ebermann et al. 73. Introduction | 43 Vestibular phenotypes In addition to the various hearing characteristics, a vestibular phenotype can also be a characteristic of a genetic dysfunction. Mutations in ten genes are known to cause vestibular problems in addition to hearing impairments, all of which have been described above. For severe prelingual hearing impairments, vestibular problems have been reported for DFNB2 (MYO7A) 27, DFNB36 (ESPN) 30 and DFNB37 (MYO6) 29. For progressive hearing impairments, hyporeflexia and areflexia were found in DFNB84A (PTPRQ) 49 and DFNB102 (CLIC5) 51. Flat to downsloping audiogram configurations can be accompanied by vestibular dysfunction in DFNB18B (OTOG), DFNB84B (OTOGL) and DFNB25 (GRXCR1) 35, 37. In DFNB4 (SLC26A4), taking into account the anatomical abnormalities, vestibular dysfunction can be expected to occur 58, 60, and for auditory neuropathies, central vestibular dysfunction is reported in DFNB59 (PJVK) 73. Essence of genotype- phenotype correlations Many novel deafness- associated genes have been described in the literature, but the articles often lack important additional information, particularly those characteristics that are essential to providing proper counseling to patients regarding future expectations for their hearing impairment when the causative genetic defects are identified. Therefore, we advocate for the inclusion of a thorough phenotype description in all articles about the genetics of hearing loss. Such a description should include the age of onset, severity, progression of hearing impairment, audiogram configuration and vestibular function according to GENDEAF recommendations. The latter, especially, can be a distinctive feature. One should keep in mind that vestibular problems are not always reported by patients, who often compensate well for vestibular dysfunction 74. Extensive vestibular examination including vHIT, caloric testing, and otolith function tests should, therefore, be routinely performed for a number of patients in genotype-phenotype correlation descriptions. 44 | Chapter 1 Conclusion Counseling of patients with hereditary hearing impairment is important because it informs them about the prognosis of their hearing loss, and on the implications for their progeny. This review of different types of autosomal recessively inherited nonsyndromic hearing impairment can serve as a reference for this purpose. Many articles on the identification of novel deafness- associated genes often lack exact and thorough data on clinical features. Therefore, we recommend a thorough genotype-phenotype description be included in such articles. Introduction | 45 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Kochhar A, Hildebrand MS, Smith RJ. Clinical aspects of hereditary hearing loss. 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Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy. Nature genetics. 2006; 38(7):770-8. Ebermann I, Walger M, Scholl HP, et al. Truncating mutation of the DFNB59 gene causes cochlear hearing impairment and central vestibular dysfunction. Human mutation. 2007;28(6):571-7. Street VA, Kallman JC, Strombom PD, et al. Vestibular function in families with inherited autosomal dominant hearing loss. J Vestib Res-Equilib Orientat. 2008;18(1):51-8. Introduction | 49 50 | Chapter 1 Supplemental table 1 Phenotype characteristics of prelingual, severe to profound autosomal recessive hearing impairment. Locus Gene Onset DFNB2 [1-5] MYO7A Variable (congenital – 16 years) Severe-profound Severity Flat Audiogram configuration DFNB3 [6-17] MYO15A Congenital Severe-profound Downsloping DFNB6 [18-22] TMIE Congenital/ prelingual Severe-profound All frequencies DFNB7/11 TMC1 [23-34] Congenital, 1 postlingual [35] Severe-profound Flat- U shaped - downsloping DFNB18A USH1C [36-38] Prelingual Severe-profound DFNB22 [39-42] OTOA Prelingual Severe-profound DFNB23 [43-45] PCDH15 Prelingual Severe-profound DFNB24 [46-48] RDX Congenital, prelingual Severe-profound Flat- shallow U shape DFNB28 [49-51] TRIOBP Congenital/ prelingual Profound All frequencies DFNB29 [52-57] CLDN14 Congenital, prelingual Severe-profound Flat- gently downsloping DFNB31 [58-60] WHRN Congenital, prelingual Profound DFNB35 [61-65] ESRRB Prelingual Severe-profound Flat DFNB36 [66, 67] ESPN Prelingual Profound Flat DFNB37 [68] MYO6 Congenital Profound DFNB39 [69, 70] HGF Prelingual Severe-profound Downsloping DFNB42 [71, 72] ILDR2 Congenital, prelingual Severe-profound Flat- steeply downsloping DFNB48 [73, 74] CIB2 Congenital Severe-profound Flat DFNB49 [75-78] MARVELD2 Congenital Severe-profound Flat-downsloping Flat Introduction | 51 Vestibular involvement Geographic occurrence Additional information Variable (some do have vestibular dysfunction). Pakistan, Tunisia, Iran, China Atypical Usher is also reported (mild retinitis pigmentosa, clinically no problems). No vestibular problems. Indonesia, India, Pakistan, Turkey, Iran, Brazil, Tunisia, Qatar, the Netherlands* Can be moderate in the low frequencies. 1 family delayed motor milestones. Further NA India, Pakistan, Jordan, Turkey, Taiwan No vestibular problems. The Netherlands, Greece, Turkey, Tunisia, Lebanon, Jordan, India, Pakistan, China, Sudan, Iran NA India, China NA Palestine, Pakistan NA Pakistan, New Foundland (Canada) NA Pakistan, Iran, India NA Turkey, India, Palestine, Pakistan NA Pakistan, Morocco, Greece Progressive in family with post lingual onset Variability intrafamiliar. Can be moderate in the low frequencies Palestine, Tunisia NA Pakistan, Tunisia, Czech, Turkey 1/3 families shows areflexia Pakistan, Morocco during caloric testing Abnormal (2/9), others not tested Pakistan NA Pakistan, India NA Pakistan, Iran, the Netherlands* Variable other findings Not progressive Can be moderate in the low frequencies Pakistan, Curacao* NA Pakistan, Czech (Roma) Progressive from severe to profound 52 | Chapter 1 Supplemental table 1 Continued. Locus Gene Onset Severity Audiogram configuration DFNB53 [79] COL11A2 Prelingual Profound Flat DFNB61 [80] SLC26A5 Congenital Severe-profound Flat DFNB63 [81-86] COMT2 Congenital Severe-profound Gently- steeply downsloping DFNB67 [40, 87, 88] LHFPL5/ LRTOMT Congenital, prelingual Severe- profound DFNB66 [89] DCDC2 Congenital Profound DFNB70 [90] PNPT1 Prelingual Severe-profound DFNB73 [91] BSND DFNB74 [92, 93] MSRB3 DFNB79 [94-97] Severe-profound Flat Prelingual Profound Flat TPRN Prelingual Severe- profound Flat-steeply downsloping DFNB82 [98, 99] GPSM2 Prelingual Severe- profound Gently downsloping DFNB86 [100, 101] TBC1D24 Prelingual Profound DFNB89 KARS [102, 103] Prelingual Moderateprofound DFNB94 [104] NARS2 Prelingual, congenital Profound DFNB97 [105] MET Prelingual Severe- profound DFNB98 [106] TSPEAR Prelingual Profound DFNB99 [107] TMEM132E Prelingual Severe- profound DFNB102 EPS8 [108] Congenital Profound [109] Congenital Profound FAM65B *marks the origin of a family where mutations are found, but which is not published Flat-gently downsloping Flat Introduction Vestibular involvement Geographic occurrence NA Iran NA Caucasian (USA) NA Iran, Turkey, Pakistan, Morocco, Tunisia NA Turkey, Palestine, India, Pakistan NA Tunisia NA Morocco NA Pakistan NA Pakistan NA The Netherlands, Morocco, Pakistan NA Palestine, Turkey NA Pakistan NA Pakistan No vestibular problems Pakistan No vestibular problems Pakistan Iran No vestibular problems China NA Algeria No vestibular problems Turkey | 53 Additional information Can be moderate in the low frequencies Subclinical renal metabolic changes Progression reported 54 | Chapter 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 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Proceedings of the National Academy of Sciences of the United States of America. 2014;111(27):9864-8. 60 | Chapter 1 Supplemental table 2 Autosomal recessive types of hearing impairment with specific phenotypes characteristics. Locus Gene Onset Severity Audiogram configuration DFNB1 GJB2/GJB6 Congenital, prelingual Mild-profound (variable) Flat- downsloping (variable) DFNB4 [1-5] SLC26A4 Mostly prelingual Mild- profound DFNB8/10 [6-18] TMPRSS3 Prelingual (DFNB10) and postlingual (DFNB8) Moderateprofound Steeply downsloping, progression until flat DFNB12 [19-27] CDH23 Congenital, prelingual Moderateprofound Flat- steeply downsloping DFNB15/72/95 GIPC3 [28-33] Prelingual Moderateprofound Flat- U shaped- gently downsloping DFNB16 [34-37] STRC Early childhood Mostly mildDownsloping moderate, can be severe - profound DFNB18B [38, 39] OTOG Prelingual Mild- moderate Flat-gently downsloping DFNB21 [40-46] TECTA Prelingual Moderate- severe Flat- shallow U shape DFNB25 [47] GRXCR1 Congenital Moderateprofound Flat- gently downsloping DFNB30 [48] MYO3A Second decade Moderate- severe Downsloping DFNB49 [49] BDP1 Post lingual Moderate- severe Gently downsloping DFNB76 [50] SYN4 Early onset (congenital – 6 years) Severe by adulthood (initial level is not reported) Steeply downsloping DFNB77 [18, 51, 52] LOXHD1 Reported prelingual and post lingual Moderateprofound Steeply sloping in low frequencies, flat in mid and high frequencies Introduction | 61 Vestibular involvement Number of Geographic occurrence families in literature Additional information No consensus >100 Most common form of ARNSHI Progressive and stable types are reported Vestibular dysfunction, like >100 complaints of vertigo, are reported Caucasian, Asia Temporal bone abnormalities. Great variability. Fluctuation and/ or progression. Hyporeflexia and >30 hyperreflexia were detected during electronystagmography, although the overall prevalence was not above change level. 8: Pakistan, Turkey, UK, Germany, the Netherlands, Korea 10: Palestine, Tunisia, Turkey, Pakistan, Greece, Spain, the Netherlands, Italy, India Progressive Normal vestibular function >40 Pakistan, Japan, Germany, Progression is reported. African America, European, the Netherlands, India, North America, Korea No vestibular problems reported 11 The Netherlands*, Turkey, Pakistan, Saudi Arabia, India. No vestibular problems reported 20 French, Pakistan, Caucasian, Non progressive Asian, Middle East Delayed motor milestones, vestibular hypofunction 2 The Netherlands, Spain. Might be slightly progressive later in life NA 9 Lebanon, Pakistan, Iran, Korea Non progressive, unilateral HI reported in one patient Vestibular dysfunction has been reported. 4 The Netherlands, Pakistan Can be progressive No vestibular problems reported 1 Jewish family (Iraq) Progressive NA 1 Qatar Mildly progressive NA 2 Iraqi (Jew) Progressive No vestibular problems reported 6 Iran, Turkey, Askenazi Jews, Progressive Qatar, the Netherlands* 62 | Chapter 1 Supplemental table 2 Autosomal recessive types of hearing impairment with specific phenotypes characteristics. Onset Severity Audiogram configuration DFNB84A PTPRQ [53-55] Prelingual Moderateprofound Flat DFNB84B OTOGL [39, 56] Prelingual Mild- moderate Flat- gently downsloping DFNB91 [57] SERPINB6 Post lingual Moderate- severe Flat- steeply downsloping DFNB93 [58, 59] CABP2 Prelingual Moderate- severe Flat- shallow U shape DFNB101 GRXCR2 [60] Early childhood moderate Gently downsloping DFNB103 CLIC5 [61] Prelingual [62] GJB3 Early childhood Severe Flat [63] USH1G Early childhood Moderate- severe Downsloping Locus Gene Downsloping *marks the origin of a family where mutations are found, but which is not published. 1. 2. 3. 4. 5. 6. 7. 8. 9. Berrettini S, Forli F, Bogazzi F, et al. Large vestibular aqueduct syndrome: audiological, radiological, clinical, and genetic features. American journal of otolaryngology. 2005;26(6):363-71. Wang Q J, Zhao YL, Rao SQ, et al. A distinct spectrum of SLC26A4 mutations in patients with enlarged vestibular aqueduct in China. Clinical genetics. 2007;72(3):245-54. Albert S, Blons H, Jonard L, et al. SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations. European journal of human genetics : EJHG. 2006;14(6):773-9. Busi M, Castiglione A, Taddei Masieri M, et al. Novel mutations in the SLC26A4 gene. International journal of pediatric otorhinolaryngology. 2012;76(9):1249-54. Miyagawa M, Nishio SY, Usami S, et al. Mutation spectrum and genotype-phenotype correlation of hearing loss patients caused by SLC26A4 mutations in the Japanese: a large cohort study. Journal of human genetics. 2014;59(5):262-8. Weegerink NJ, Schraders M, Oostrik J, et al. Genotype-phenotype correlation in DFNB8/10 families with TMPRSS3 mutations. Journal of the Association for Research in Otolaryngology : JARO. 2011;12(6):753-66. Ahmed ZM, Li XC, Powell SD, et al. Characterization of a new full length TMPRSS3 isoform and identification of mutant alleles responsible for nonsyndromic recessive deafness in Newfoundland and Pakistan. BMC medical genetics. 2004;5:24. Ben-Yosef T, Wattenhofer M, Riazuddin S, et al. Novel mutations of TMPRSS3 in four DFNB8/B10 families segregating congenital autosomal recessive deafness. Journal of medical genetics. 2001;38(6): 396-400. Elbracht M, Senderek J, Eggermann T, et al. Autosomal recessive postlingual hearing loss (DFNB8): compound heterozygosity for two novel TMPRSS3 mutations in German siblings. Journal of medical genetics. 2007;44(6):e81. Introduction Vestibular involvement Number of Geographic occurrence families in literature | 63 Additional information Hypofunction and complete 4 areflexia are reported The Netherlands, Palestine, Morocco Delayed motor milestones, vestibular hypofunction 3 The Netherlands, Turkey, France. No vestibular problems reported 1 Turkey Progression reported NA 3 Iran Stable No vestibular problems reported 1 Pakistan Progressive Vestibular areflexia and 1 associated balance problems Turkey Highly progressive vestibular and hearing loss. No vestibular problems reported 2 China Digenic inheritance is also reported Normal vestibular function 1 The Netherlands Progressive 10. 11. 12. 13. 14. 15. 16. 17. 18. Progressive, variability within the family Masmoudi S, Antonarakis SE, Schwede T, et al. Novel missense mutations of TMPRSS3 in two consanguineous Tunisian families with non-syndromic autosomal recessive deafness. Human mutation. 2001;18(2):101-8. Scott HS, Kudoh J, Wattenhofer M, et al. Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness. Nature genetics. 2001;27(1):59-63. Veske A, Oehlmann R, Younus F, et al. Autosomal recessive non-syndromic deafness locus (DFNB8) maps on chromosome 21q22 in a large consanguineous kindred from Pakistan. Human molecular genetics. 1996;5(1):165-8. Wattenhofer M, Di Iorio MV, Rabionet R, et al. Mutations in the TMPRSS3 gene are a rare cause of childhood nonsyndromic deafness in Caucasian patients. Journal of molecular medicine (Berlin, Germany). 2002;80(2):124-31. Wattenhofer M, Sahin-Calapoglu N, Andreasen D, et al. A novel TMPRSS3 missense mutation in a DFNB8/10 family prevents proteolytic activation of the protein. Human genetics. 2005;117(6):528-35. Hutchin T, Coy NN, Conlon H, et al. Assessment of the genetic causes of recessive childhood nonsyndromic deafness in the UK - implications for genetic testing. Clinical genetics. 2005;68(6):506-12. Bonne-Tamir B, DeStefano AL, Briggs CE, et al. Linkage of congenital recessive deafness (gene DFNB10) to chromosome 21q22.3. American journal of human genetics. 1996;58(6):1254-9. Walsh T, Abu Rayan A, Abu Sa’ed J, et al. Genomic analysis of a heterogeneous Mendelian phenotype: multiple novel alleles for inherited hearing loss in the Palestinian population. Human genomics. 2006;2(4):203-11. Vozzi D, Morgan A, Vuckovic D, et al. Hereditary hearing loss: a 96 gene targeted sequencing protocol reveals novel alleles in a series of Italian and Qatari patients. Gene. 2014;542(2):209-16. 64 | Chapter 1 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Ammar-Khodja F, Faugere V, Baux D, et al. Molecular screening of deafness in Algeria: high genetic heterogeneity involving DFNB1 and the Usher loci, DFNB2/USH1B, DFNB12/USH1D and DFNB23/ USH1F. European journal of medical genetics. 2009;52(4):174-9. Astuto LM, Bork JM, Weston MD, et al. CDH23 mutation and phenotype heterogeneity: a profile of 107 diverse families with Usher syndrome and nonsyndromic deafness. American journal of human genetics. 2002;71(2):262-75. Bork JM, Peters LM, Riazuddin S, et al. Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23. American journal of human genetics. 2001;68(1):26-37. Chaib H, Place C, Salem N, et al. Mapping of DFNB12, a gene for a non-syndromal autosomal recessive deafness, to chromosome 10q21-22. Human molecular genetics. 1996;5(7):1061-4. Pennings RJ, Topsakal V, Astuto L, et al. Variable clinical features in patients with CDH23 mutations (USH1D-DFNB12). Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology. 2004;25(5):699-706. Schultz JM, Bhatti R, Madeo AC, et al. Allelic hierarchy of CDH23 mutations causing non-syndromic deafness DFNB12 or Usher syndrome USH1D in compound heterozygotes. Journal of medical genetics. 2011;48(11):767-75. Wagatsuma M, Kitoh R, Suzuki H, et al. Distribution and frequencies of CDH23 mutations in Japanese patients with non-syndromic hearing loss. Clinical genetics. 2007;72(4):339-44. Miyagawa M, Nishio SY, Usami S. Prevalence and clinical features of hearing loss patients with CDH23 mutations: a large cohort study. PloS one. 2012;7(8):e40366. Ganapathy A, Pandey N, Srisailapathy CR, et al. Non-syndromic hearing impairment in India: high allelic heterogeneity among mutations in TMPRSS3, TMC1, USHIC, CDH23 and TMIE. PloS one. 2014; 9(1):e84773. Chen A, Wayne S, Bell A, et al. New gene for autosomal recessive non-syndromic hearing loss maps to either chromosome 3q or 19p. American journal of medical genetics. 1997;71(4):467-71. Ain Q, Nazli S, Riazuddin S, et al. The autosomal recessive nonsyndromic deafness locus DFNB72 is located on chromosome 19p13.3. Human genetics. 2007;122(5):445-50. Charizopoulou N, Lelli A, Schraders M, et al. Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human. Nature communications. 2011;2:201. Rehman AU, Gul K, Morell RJ, et al. Mutations of GIPC3 cause nonsyndromic hearing loss DFNB72 but not DFNB81 that also maps to chromosome 19p. Human genetics. 2011;130(6):759-65. Sirmaci A, Edwards YJ, Akay H, et al. Challenges in whole exome sequencing: an example from hereditary deafness. PloS one. 2012;7(2):e32000. Ramzan K, Al-Owain M, Allam R, et al. Homozygosity mapping identifies a novel GIPC3 mutation causing congenital nonsyndromic hearing loss in a Saudi family. Gene. 2013;521(1):195-9. Francey LJ, Conlin LK, Kadesch HE, et al. Genome-wide SNP genotyping identifies the Stereocilin (STRC) gene as a major contributor to pediatric bilateral sensorineural hearing impairment. American journal of medical genetics Part A. 2012;158A(2):298-308. Verpy E, Masmoudi S, Zwaenepoel I, et al. Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus. Nature genetics. 2001;29(3):345-9. Villamar M, del Castillo I, Valle N, et al. Deafness locus DFNB16 is located on chromosome 15q13-q21 within a 5-cM interval flanked by markers D15S994 and D15S132. American journal of human genetics. 1999;64(4):1238-41. Campbell DA, McHale DP, Brown KA, et al. A new locus for non-syndromal, autosomal recessive, sensorineural hearing loss (DFNB16) maps to human chromosome 15q21-q22. Journal of medical genetics. 1997;34(12):1015-7. Schraders M, Ruiz-Palmero L, Kalay E, et al. Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment. American journal of human genetics. 2012;91(5):883-9. Oonk AM, Leijendeckers JM, Huygen PL, et al. Similar phenotypes caused by mutations in OTOG and OTOGL. Ear Hear. 2014;35(3):e84-91. Introduction 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. | 65 Mustapha M, Weil D, Chardenoux S, et al. An alpha-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21. Human molecular genetics. 1999;8(3):409-12. Naz S, Alasti F, Mowjoodi A, et al. Distinctive audiometric profile associated with DFNB21 alleles of TECTA. Journal of medical genetics. 2003;40(5):360-3. Meyer NC, Alasti F, Nishimura CJ, et al. Identification of three novel TECTA mutations in Iranian families with autosomal recessive nonsyndromic hearing impairment at the DFNB21 locus. American journal of medical genetics Part A. 2007;143A(14):1623-9. Alasti F, Sanati MH, Behrouzifard AH, et al. A novel TECTA mutation confirms the recognizable phenotype among autosomal recessive hearing impairment families. International journal of pediatric otorhinolaryngology. 2008;72(2):249-55. Sagong B, Park R, Kim YH, et al. Two novel missense mutations in the TECTA gene in Korean families with autosomal dominant nonsyndromic hearing loss. Annals of clinical and laboratory science. 2010;40(4):380-5. Diaz-Horta O, Duman D, Foster J, 2nd, et al. Whole-exome sequencing efficiently detects rare mutations in autosomal recessive nonsyndromic hearing loss. PloS one. 2012;7(11):e50628. Dodson KM, Georgolios A, Barr N, et al. Etiology of unilateral hearing loss in a national hereditary deafness repository. American journal of otolaryngology. 2012;33(5):590-4. Schraders M, Lee K, Oostrik J, et al. Homozygosity mapping reveals mutations of GRXCR1 as a cause of autosomal-recessive nonsyndromic hearing impairment. American journal of human genetics. 2010;86(2):138-47. Walsh T, Walsh V, Vreugde S, et al. From flies’ eyes to our ears: mutations in a human class III myosin cause progressive nonsyndromic hearing loss DFNB30. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(11):7518-23. Girotto G, Mezzavilla M, Abdulhadi K, et al. Consanguinity and hereditary hearing loss in Qatar. Human heredity. 2014;77(1-4):175-82. Horn HF, Brownstein Z, Lenz DR, et al. The LINC complex is essential for hearing. The Journal of clinical investigation. 2013;123(2):740-50. Grillet N, Schwander M, Hildebrand MS, et al. Mutations in LOXHD1, an evolutionarily conserved stereociliary protein, disrupt hair cell function in mice and cause progressive hearing loss in humans. American journal of human genetics. 2009;85(3):328-37. Edvardson S, Jalas C, Shaag A, et al. A deleterious mutation in the LOXHD1 gene causes autosomal recessive hearing loss in Ashkenazi Jews. American journal of medical genetics Part A. 2011;155A(5):1170-2. Schraders M, Oostrik J, Huygen PL, et al. Mutations in PTPRQ are a cause of autosomal-recessive nonsyndromic hearing impairment DFNB84 and associated with vestibular dysfunction. American journal of human genetics. 2010;86(4):604-10. Shahin H, Rahil M, Abu Rayan A, et al. Nonsense mutation of the stereociliar membrane protein gene PTPRQ in human hearing loss DFNB84. Journal of medical genetics. 2010;47(9):643-5. Shahin H, Walsh T, Rayyan AA, et al. Five novel loci for inherited hearing loss mapped by SNP-based homozygosity profiles in Palestinian families. European journal of human genetics: EJHG. 2010; 18(4):407-13. Yariz KO, Duman D, Seco CZ, et al. Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss. American journal of human genetics. 2012;91(5):872-82. Sirmaci A, Erbek S, Price J, et al. A truncating mutation in SERPINB6 is associated with autosomalrecessive nonsyndromic sensorineural hearing loss. American journal of human genetics. 2010;86(5): 797-804. Tabatabaiefar MA, Alasti F, Shariati L, et al. DFNB93, a novel locus for autosomal recessive moderateto-severe hearing impairment. Clinical genetics. 2011;79(6):594-8. Schrauwen I, Helfmann S, Inagaki A, et al. A mutation in CABP2, expressed in cochlear hair cells, causes autosomal-recessive hearing impairment. American journal of human genetics. 2012;91(4):636-45. Imtiaz A, Kohrman DC, Naz S. A Frameshift Mutation in GRXCR2 Causes Recessively Inherited Hearing Loss. Human mutation. 2014;35(5):618-24. 66 | Chapter 1 61. 62. 63. Seco CZ, Oonk AM, Dominguez-Ruiz M, et al. Progressive hearing loss and vestibular dysfunction caused by a homozygous nonsense mutation in CLIC5. European journal of human genetics : EJHG. 2014. Liu XZ, Xia XJ, Xu LR, et al. Mutations in connexin31 underlie recessive as well as dominant non-syndromic hearing loss. Human molecular genetics. 2000;9(1):63-7. Oonk AM, van Huet RA, Leijendeckers JM, et al. Nonsyndromic hearing loss caused by USH1G mutations: widening the USH1G disease spectrum. Ear Hear. 2015;36(2):205-11. Introduction | 67 68 | Chapter 1 Supplemental table 3 Phenotype characteristics of auditory neuropathies. Locus Gene Onset Severity Audiogram configuration DFNB9 [1-17] OTOF Congenital, prelingual Severe- profound Flat- U shaped- downsloping DFNB59 [18-25] PJVK Congenital, prelingual Moderate- profound Flat- gently downsloping 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Chaib H, Place C, Salem N, et al. A gene responsible for a sensorineural nonsyndromic recessive deafness maps to chromosome 2p22-23. Human molecular genetics. 1996;5(1):155-8. Leal SM, Apaydin F, Barnwell C, et al. A second middle eastern kindred with autosomal recessive nonsyndromic hearing loss segregates DFNB9. European journal of human genetics : EJHG. 1998;6(4):341-4. Yasunaga S, Grati M, Cohen-Salmon M, et al. A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nature genetics. 1999;21(4):363-9. Adato A, Raskin L, Petit C, et al. Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus. European journal of human genetics : EJHG. 2000;8(6):437-42. Yasunaga S, Grati M, Chardenoux S, et al. OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9. American journal of human genetics. 2000;67(3):591-600. Houseman MJ, Jackson AP, Al-Gazali LI, et al. A novel mutation in a family with non-syndromic sensorineural hearing loss that disrupts the newly characterised OTOF long isoforms. Journal of medical genetics. 2001;38(8):E25. Migliosi V, Modamio-Hoybjor S, Moreno-Pelayo MA, et al. Q829X, a novel mutation in the gene encoding otoferlin (OTOF), is frequently found in Spanish patients with prelingual non-syndromic hearing loss. Journal of medical genetics. 2002;39(7):502-6. Rodriguez-Ballesteros M, del Castillo FJ, Martin Y, et al. Auditory neuropathy in patients carrying mutations in the otoferlin gene (OTOF). Human mutation. 2003;22(6):451-6. Varga R, Kelley PM, Keats BJ, et al. Non-syndromic recessive auditory neuropathy is the result of mutations in the otoferlin (OTOF) gene. Journal of medical genetics. 2003;40(1):45-50. Gallo-Teran J, Megia Lopez R, Morales-Angulo C, et al. [Evaluation of a family with sensorineural hearing loss due to the Q829X mutation in the OTOF gene]. Acta otorrinolaringologica espanola. 2004;55(3):120-5. Tekin M, Akcayoz D, Incesulu A. A novel missense mutation in a C2 domain of OTOF results in autosomal recessive auditory neuropathy. American journal of medical genetics Part A. 2005;138(1):6-10. Gallo-Teran J, Morales-Angulo C, Rodriguez-Ballesteros M, et al. [Prevalence of the 35delG mutation in the GJB2 gene, del (GJB6-D13S1830) in the GJB6 gene, Q829X in the OTOF gene and A1555G in the mitochondrial 12S rRNA gene in subjects with non-syndromic sensorineural hearing impairment of congenital/childhood onset]. Acta otorrinolaringologica espanola. 2005;56(10):463-8. Varga R, Avenarius MR, Kelley PM, et al. OTOF mutations revealed by genetic analysis of hearing loss families including a potential temperature sensitive auditory neuropathy allele. Journal of medical genetics. 2006;43(7):576-81. Choi BY, Ahmed ZM, Riazuddin S, et al. Identities and frequencies of mutations of the otoferlin gene (OTOF) causing DFNB9 deafness in Pakistan. Clinical genetics. 2009;75(3):237-43. Introduction Vestibular involvement NA, central vestibular dysfunction 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. | 69 Geographic occurrence Additional information France, Spain, UK, Italy, Germany, Austria, USA, Israel, Turkey, Lebanon, Palestine, Libya, UAE, Iran, Pakistan, Israel, Argentina, Brazil, Mexico, Cuba, Colombia, China, Japan, Taiwan. Temperature sensitive Pakistan, Israel, Morocco, Iran, Turkey, China Can be moderate in the low frequencies Stable and progressive have been reported. Marlin S, Feldmann D, Nguyen Y, et al. Temperature-sensitive auditory neuropathy associated with an otoferlin mutation: Deafening fever! Biochemical and biophysical research communications. 2010;394(3):737-42. Mahdieh N, Shirkavand A, Rabbani B, et al. Screening of OTOF mutations in Iran: a novel mutation and review. International journal of pediatric otorhinolaryngology. 2012;76(11):1610-5. Matsunaga T, Mutai H, Kunishima S, et al. A prevalent founder mutation and genotype-phenotype correlations of OTOF in Japanese patients with auditory neuropathy. Clinical genetics. 2012;82(5):425-32. Delmaghani S, del Castillo FJ, Michel V, et al. Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy. Nature genetics. 2006;38(7):770-8. Collin RW, Kalay E, Oostrik J, et al. Involvement of DFNB59 mutations in autosomal recessive nonsyndromic hearing impairment. Human mutation. 2007;28(7):718-23. Ebermann I, Walger M, Scholl HP, et al. Truncating mutation of the DFNB59 gene causes cochlear hearing impairment and central vestibular dysfunction. Human mutation. 2007;28(6):571-7. Hashemzadeh Chaleshtori M, Simpson MA, Farrokhi E, et al. Novel mutations in the pejvakin gene are associated with autosomal recessive non-syndromic hearing loss in Iranian families. Clinical genetics. 2007;72(3):261-3. Schwander M, Sczaniecka A, Grillet N, et al. A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2007;27(9):2163-75. Wang J, Fan YY, Wang SJ, et al. Variants of OTOF and PJVK genes in Chinese patients with auditory neuropathy spectrum disorder. PloS one. 2011;6(9):e24000. Borck G, Rainshtein L, Hellman-Aharony S, et al. High frequency of autosomal-recessive DFNB59 hearing loss in an isolated Arab population in Israel. Clinical genetics. 2012;82(3):271-6. Mujtaba G, Bukhari I, Fatima A, et al. A p.C343S missense mutation in PJVK causes progressive hearing loss. Gene. 2012;504(1):98-101. 2 Novel genotypephenotype correlations 2.1 Similar phenotypes caused by mutations in OTOG and OTOGL A.M.M. Oonk J.M. Leijendeckers P.L.M. Huygen M. Schraders M. del Campo I. del Castillo M. Tekin I. Feenstra A.J. Beynon H.P.M. Kunst A.F.M. Snik H. Kremer R.J.C. Admiraal R.J.E. Pennings Ear and Hearing 2014 2014 May-Jun;35(3):e84-91 74 | Chapter 2 Abstract Recently, OTOG and OTOGL were identified as human deafness genes. Currently, only five families are known to have autosomal recessive hearing loss based on mutations in these genes. Since the two genes code for proteins (otogelin and otogelin-like) that are strikingly similar in structure and localization in the inner ear, this study is focused on characterizing and comparing the hearing loss caused by mutations in these genes. To evaluate this type of hearing, an extensive set of audiometric and vestibular examinations was performed in the 13 patients from four families. All families show a flat to downsloping configuration of the audiogram with mild to moderate sensorineural hearing loss. Speech recognition scores remain good (>90%). Hearing loss is not significantly different in the four families and the psychophysical test results also do not differ between the families. Vestibular examinations show evidence for vestibular hyporeflexia. Since otogelin and otogelin-like are localized in the tectorial membrane, one could expect a cochlear conductive hearing loss, as was previously shown in DFNA13 (COL11A2) and DFNA8/12 (TECTA) patients. Results of psychophysical examinations, however, do not support this. Furthermore, the authors can conclude that there are no phenotypic differences between hearing loss based on mutations in OTOG or OTOGL. This phenotype description will facilitate counseling of hearing loss caused by defects in either of these two genes. Novel genotype-phenotype correlations | 75 Introduction Hearing impairment is the most common sensorineural disorder in humans and has many underlying causes e.g. infection, trauma or genetic defects. The latter are responsible for at least half of the cases with an early onset 1. For hereditary early onset hearing loss the inheritance pattern is recessive in about 80% of the cases. Different techniques are available to identify the causative gene. An example is linkage analysis in which parental consanguinity and large family size enable easier identification of causative genes 2. Since these family characteristics are not common in Western populations, it was difficult to identify new loci and genes for autosomal recessive deafness in these populations in the past. The introduction of novel screening techniques including exome sequencing has led to an increase in the number of identified genetic causes of recessively inherited hearing loss also in the Western population 3. So far, over 60 genes harboring more than 1,000 mutations were identified for non-syndromic hearing impairment 4, 5. OTOG (DFNB18B) is one of the novel human deafness- associated genes. It codes for otogelin, which is a non-collagenous protein that was found to be specific to the inner ear in mice by Cohen-Salmon et al. 6. Subsequently, Simmler et al. 7 indicated OTOG to be a mouse deafness gene. Otogelin is held responsible for binding of the otoconial membrane and cupula to the neuroepithelium. In the tectorial membrane, otogelin might be important for the interaction or stabilization of the type-A and B fibers 8. OTOG was therefore also considered to be a candidate gene for hereditary non-syndromic hearing impairment in humans 7. The gene was mapped to chromosome 11 of the human genome by Cohen- Salmon et al. 9 and we recently confirmed that mutations in OTOG cause deafness in humans 10. Shahin et al. 11 described a gene homologous to OTOG, which is called OTOGL (DFNB84B). The predicted product of this gene is otogelin-like and 33.3% of the amino acid sequence is identical to that of otogelin 12. Furthermore, otogelin-like is a component of the tectorial membrane, as is otogelin. When expression of otogl is knocked down in zebrafish, it leads to sensorineural hearing loss. Recently, we also have shown that OTOGL is a human deafness gene 12. Because of the striking similarities between otogelin and otogelin-like in terms of structure and expression, we wanted to evaluate whether the phenotypes of mutations in OTOG and OTOGL are also similar. This study presents an extensive audiometric and vestibular evaluation of the patients currently known with recessive sensorineural hearing loss caused by mutations in either OTOG or OTOGL 10, 12 . This description facilitates the identification of causative genetic defects in 76 | Chapter 2 the outpatient clinic and improves counseling of patients on prognosis and rehabilitation of their hearing loss. Patients and methods Family data Thirteen patients, from four different families were included in this study. Families A (W11-0186) and C (W00-384) are of Dutch origin, family B originates in Turkey and family D (S1778) is of Spanish origin. An autosomal recessive type of inheritance is apparent in the pedigrees, which show hearing loss in only one generation (figure 1). In family B a consanguineous marriage is present as the parents are first cousins. Figure 1 Pedigrees of families participating in this study. A square indicates a male, a circle indicates a female. A filled symbol means affected an open symbol means unaffected. Novel genotype-phenotype correlations | 77 All participants voluntarily participated in this study and informed consent was obtained from the patients or parents when the patient was a minor. This study was approved by the local medical ethical committee. All hearing impaired family members filled in a standardized questionnaire on audiovestibular symptoms and underwent ENT examination, including otoscopy and external ear inspection, to exclude external ear deformities, previous surgery and other possible causes of hearing impairment. A computed tomography (CT) scan of the temporal bone was performed in one member of family B and family C (II:3), in order to screen for possible anatomical causes of congenital hearing loss. Genetic analysis of families A and B has been described by Yariz et al. 12. Mutation analysis of OTOGL was initiated in family A and two compound heterozygous mutations were identified, a nonsense mutation (c.547C>T (p.(Arg183X))) and a splice site mutation (c.5238+5G>A). They found a mutation in a homozygous state in OTOGL in family B (c.1430delT (p.(Val477Glufs*25))). Genetic results for families C and D have been described by Schraders et al. 10. In family C a homozygous region containing OTOG was identified by homozygosity mapping and therefore, Sanger sequencing was applied to OTOG. A deletion (c.5508delC) was found in a homozygous state. This deletion is predicted to cause a frameshift and a premature stopcodon (p.(Ala1838ProfsX31)). In family D two pathogenic compound heterozygous mutations in OTOG (c.6347C>T (p.(Pro2116Leu)) and c.6559C>T (p.(Arg2187X))) were identified. Audiovestibular examination Pure tone audiometry Pure tone audiometry was performed according to current standards to determine hearing thresholds at frequencies of 0.25, 0.5, 1, 2, 4 and 8 kHz. To exclude conductive hearing impairment, air and bone conduction thresholds were determined. Speech recognition test In families A and C standard Dutch phonetically balanced word lists (NVA Dutch CVC lists, Bosman 1992) were used to measure speech recognition scores. The average of the maximum percentage correct for both ears is the maximum phoneme score. These scores were obtained from monaural performance versus intensity curves. Otoacoustic emissions (OAEs) and acoustic ref lexes OAEs were assessed in individual II:1 from family C. In all members of families A, B and C acoustic reflexes were measured contralateral and ipsilateral at 0.5, 1, 2 and 4 kHz up to the loudness discomfort level. 78 | Chapter 2 Vestibular function tests Unterberger stepping test, Romberg test, head thrust test, head shake test and smooth pursuit eye movements were used to roughly evaluate vestibular function in family A. The parents of family A did not give consent for more extensive vestibular testing. Vestibular function was evaluated in family B, C and D by electronystagmography. This involved calorics in families C and D and a velocity-step test in all three families. Calorisation was performed by bithermal (30 and 44˚C) water irrigation of the external auditory canal. The velocity step test was performed with patients seated in a rotary chair and their head anteflexed at 30˚. The chair was accelerated and when the rotatory nystagmus had subsided during constant rotation the chair was suddenly stopped. Areflexia was defined as no responses during vestibular function tests. Hyporeflexia was defined when responses are below normal ranges (i.e. velocity step test: gain < 33%, slow phase velocity < 300/s, time constant < 11 s; caloric tests: < 70/s and <100/s for cold and warm irrigation, respectively). This test was previously described by Theunissen et al. 13. Psychophysical examination In order to try to distinguish further than a mere conductive or sensorineural hearing loss we performed the following psychophysical examinations to families A and C: loudness scaling, gap detection, difference limen for frequency and speech reception in noise. The latter was measured in the soundfield, the other tests were measured with a headphone at the best performing ear. Results of loudness scaling, gap detection and difference limen for frequency were compared to results of psychophysical examination of normal hearing individuals. The data of normal hearing individuals were previously described by de Leenheer et al. and Plantinga et al. 14, 15. The results of speech reception in noise were also compared to those of presbyacusis patients 16 and normal hearing individuals. Loudness scaling was measured with the Würzburger Hörfeld Skalierung developed by Moser 17. Pure tones that ranged from threshold level (category 1) to loudness discomfort level (category 7) were presented with duration of one second. Patients were asked to rate the perceived loudness of stimuli on a scale from one to seven. Loudness scaling was performed with 0.5 kHz and 2 kHz pure tones. At these two frequencies, each stimulus level was presented four times in a random order. Gap detection was performed as described by De Leenheer et al. 15. Unfiltered white noise, octave band filtered white noise with 0.5 kHz centre frequency and with 2 kHz centre frequency were used to test the patient’s ability to perceive a period of silence between two noise bursts. These bursts are of equal duration and intensity and were presented at the most comfortable listening level (MCL). Gap widths of 0 (no gap), 2, 2.8, 4, 5.6, 8, 11.2, 16 and 22.4 ms were randomly presented. The random gap procedure was repeated four times for each type of noise. Novel genotype-phenotype correlations | 79 Difference limen for frequency (DLF) was measured at 0.5 kHz and 2 kHz with randomly emitted frequency modulated pure tones generated by an Interacoustics AC-40 audiometer. Frequency fluctuations were 0 (no fluctuation), 0.2, 0.4, 0.6, 0.8, 1, 2, 3 and 5%. All the stimuli were presented three times at the MCL. The lowest percentage of fluctuation that was detected by the patient was designated to be the DLF. The speech reception thresholds in noise (SRT), i.e. the presentation level at which a score of 50% correct is achieved for whole sentences, were determined as described by de Leenheer et al. 15. To define these SRTs, sentences in noise in the soundfield, according to Plomp and Mimpen, were used 18. The noise level was fixed at the MCL and the adaptive procedure described by Plomp and Mimpen was used to set the level of the sentences. Both speech and noise were presented via one loudspeaker facing the patient who was not wearing hearing aids. Outcome measure in this experiment was the signal-to-noise ratio (S/N ratio), which is the difference in decibels between the SRT and the noise level. The youngest member of family A did not participate in the psychophysical examinations since these tests were too difficult for his age. Statistic analyses To evaluate progression of hearing impairment linear regression analysis was performed with Prism 5.0 (Graphpad, San Diego, Ca, USA). For each measured frequency it was tested whether the regression coefficient differed significantly from 0. Loudness scaling was evaluated with linear regression analysis as well. To compare hearing loss between all four families a mean audiogram was calculated and compared by means of an unpaired Students T-test. When more than two groups were compared a one-way ANOVA was used. Results Clinical data Family A consists of three hearing impaired boys in one generation aged five to eight years at first visit. Follow-up comprised 1.8 years. In family B, four siblings were analyzed. At first visit, II:1 was 34 years old, II:4 33 years, II:6 14 years and II:7 was 10 years old. Two audiograms were available of three of the four family members each (II:1, II:6 and II:7). The time between both audiograms is about eight years. Only one audiogram was available of individual II:4. In family C, one generation consisting of four affected male siblings was analyzed. They were three to seven years old at first visit and were followed for on average 11.5 years. Family D consists of two hearing impaired siblings aged 4.9 and 6.7 years at first visit. 80 | Chapter 2 Follow-up comprised on average 21 years. The onset of hearing loss is potentially prelingual in families A, C and D. One member (AII:3) of family A failed neonatal hearing screening. Subsequently, the two older brothers were diagnosed with hearing loss at the age of two (II:2) and three (II:1) years. Speech development was A dB B dB -10 0 -10 0 20 20 40 40 60 60 E 80 80 dB 100 100 -10 0 120 120 140 .25 140 .25 .5 1 2 4 8 kHz Combined A B C D 20 .5 1 2 4 40 8 kHz 60 C dB -10 0 -10 0 20 20 40 40 60 60 80 80 100 100 100 120 140 .25 120 120 140 .25 140 .25 .5 1 2 4 8 kHz 80 D dB .5 1 2 4 -10 0 -10 0 20 20 40 40 60 60 80 80 100 100 120 120 140 .25 140 .25 1 2 4 8 kHz G dB II: 1 .5 1 8 kHz F dB .5 2 4 8 kHz II: 2 .5 1 2 4 8 kHz Figure 2 A. Audiogram with the average hearing loss of all affected family members of family A. Standard deviations are indicated by vertical lines. B. All affected family members of family B. C. Family C. D. Family D. E. All four mean audiograms combined. F. All audiograms (mean of both ears) of individual II.1 of family D combined (6.7-24 years of age). G. All audiograms (mean of both ears) individual II.2 of family D combined (4.9-30.4 years of age). Novel genotype-phenotype correlations | 81 delayed in families C (II:1, II:2 and II:4) and D (both affected members). Physical examination did not demonstrate any dysmorphic features. CT scans of the temporal bone in subjects of family B and family C did not show any abnormalities. Audiometric results Overall, affected individuals in family A-D have a mild to moderate sensorineural hearing loss with a configuration that is flat to gently downsloping from the lowto the mid-frequencies 19. This is equivalent to the results recently presented by Bonnet et al. 20 for a patient with mutations in OTOGL. Figure 2A-E shows the mean audiograms for each family separately, as well as combined. Hearing loss varies between 25 and 65 dB HL, depending on the frequency. Hearing thresholds do not significantly differ between the four families. Affected individuals of families A, B and C do not report progression of hearing loss. Longitudinal regression analysis indeed does not show progression. In family D, significant increase of thresholds at frequencies 0.25 kHz (0.63 dB/yr), 1 kHz (0.53 dB/yr), 2 kHz (0.85 dB/yr) and 4 kHz (1.17 dB/ yr) is seen in II:1 and at 1 kHz (0.35 dB/yr) and 2 kHz (0.50 dB/yr) in II:2 (figure 2F-G). This progression predominantly occurs after the age of twenty years. Cross-sectional analysis of speech recognition scores shows that speech recognition remains stable and above the 90% score. OAEs could not be detected in II:1 from family C. Average reflex thresholds for normal hearing individuals and patients with a hearing loss up to 50 dB HL are 85 dB HL. There is an interindividual standard deviation of 7 dB 21. Reflexes within the normal range were detected in all individuals, except for II:4 of family C. His reflex thresholds were beyond the loudness discomfort level. Psychophysical results of both Dutch families Individuals of family A had an average age of 9.3 years and those in family C 16.5 years at psychophysical examination. Mean results for loudness scaling experiments at 0.5 and 2 kHz are depicted in figure 3. For 0.5 kHz, the curves of loudness growth run almost parallel to or slightly steeper than those of normal hearing individuals. For 2 kHz, the curves are clearly steeper in the affected individuals as compared to those of controls. This suggests recruitment at 2 kHz, which holds for both families. The slopes for both frequencies do not differ significantly between both families (0.5 kHz p=0.62; 2 kHz p=0.57). The results of average gap detection for unfiltered white noise stimuli and for filtered white noise stimuli with 0.5 kHz and 2 kHz centre frequencies are displayed in figure 4. Results of the youngest family member of family C (II:4, aged 13.9) were qualified as unreliable since he failed to understand the explanation of the test and therefore the results were excluded. For unfiltered white noise stimuli, the average gap width was a little higher for patients than for normal hearing 82 | Chapter 2 B 0.5 kHz 2 kHz 7 7 6 6 Loudness scale Loudness scale A 5 4 3 2 1 5 4 3 2 1 0 0 0 20 40 60 80 100 120 0 20 40 db HL 60 80 100 120 db HL Figure 3 A. Loudness scaling at 0.5 kHz, dotted line indicates mean results of normal hearing individuals, dashed lines represent the results of family A, solid lines indicate family C. B. Similar results of loudness scaling at 2 kHz. white noise 0.5 kHz 10 2 kHz unreliable Gap detection (ms) 15 5 al or m C N ge ra Av e ge ra Av e C A I I. 4 3 C I I. 2 I I. C I I. 1 C 2 I I. A A I I. 1 0 Subjects Figure 4 Gap detection measured in milliseconds (ms) for unfiltered white noise stimuli and for filtered white noise stimuli with 0.5 kHz and 2 kHz centre frequencies. Mean results for families A and C compared to those of normal hearing individuals. individuals (one way ANOVA, F(2,20)=280, p<0.001). This was more prominent in family A compared to family C, although the two families did not differ significantly from one another (p=0.30). For filtered white noise stimuli with 0.5 kHz centre frequency the average values for families A and C were both smaller than for the average normal hearing individual (one way ANOVA, F(2,18)=0.82, p=0.45). On the other hand, for the filtered white noise stimuli with 2 kHz centre frequency the average value for family A was higher than for the average normal hearing individual and the average value for family C was lower than for the Novel genotype-phenotype correlations | 83 1.0 0.8 DLF (%) 0.5 kHz 0.6 2 kHz 0.4 0.2 al N or m C ge ge ra Av e ra Av e C C I I. I I. A 4 3 2 I I. I I. 1 C A A C I I. I I. 1 2 0.0 Subjects Figure 5 Difference Limen for Frequency (DLF) measured in percentage (%) for 0.5 kHz and 2 kHz. Mean results for families A and C and the results for normal hearing individuals are presented. average normal hearing individual (one way ANOVA. F(2,19)=0.12, p=0.89). Between both families the differences were not significant (0.5 kHz p=0.67; 2 kHz p=0.43). The average results for the difference limen for frequency experiments with 0.5 kHz and 2 kHz stimuli are compared between the affected individuals and controls in figure 5. Individuals with normal hearing achieve an average DLF of 0.5% in response to a 0.5 kHz tone. The DLF averages of families A and C were slightly 2 S/N ratio 0 -2 -4 ra g Av e A er ag e C N o Co r m Pr rr a es ec by l te cu d pr si s es by cu si s 4 3 I I. Av e C I I. I I. 2 C C I I. 1 C 2 I I. A A I I. 1 -6 Subjects Figure 6 SRT in noise in signal/ noise ratio. Mean results for families A and C compared to those for normal hearing individuals, presbyacusis patients and presbyacusis corrected for audibility. 84 | Chapter 2 higher at 0.5 kHz than normal hearing individuals. As for the 2 kHz stimuli, the average DLF values of families A and C were clearly higher than the average value of normal hearing individuals. Again, both families did not differ from one another (0.5 kHz, p=0.34; 2 kHz, p=1). The average values of speech reception thresholds in noise are higher, i.e. worse, for both families than for the average normal hearing individual. This is more prominent in family A, but does not differ significantly from family C (p=0.07) (figure 6). Vestibular examination In family A, individual II:1 reported delayed motor development. He could only roll over at 12 months of age and started walking after 21 months. The head thrust test showed signs of hyporeflexia of the right vestibulum. In family C, delayed motor development was reported for two boys. Individuals II:2 and II:4 were able to sit at 12 months, could stand at 14 months, crawled at 12 and 11 months respectively and walked after 18 and over 24 months, respectively. The rotatory tests revealed hyporeflexia and calorisation showed bilateral weakness in all affected males of this family. Both members of family D underwent vestibular testing and calorisation showed a bilateral deficit. In family B one member (II:7) underwent calorisation and showed vestibular hypofunction of the left vestibulum. Discussion Recently, OTOG and OTOGL were identified as novel human deafness genes and their striking similarities in protein structure and localization in the tectorial membrane were emphasized 10, 12. In the present study, the phenotypic characteristics of two families with OTOG mutations and two families with OTOGL mutations are evaluated to compare both phenotypes. All affected family members show a mild to moderate hearing loss and a flat to gently downsloping audiogram, no differences between the four families were noted. Mild progression may be seen in families with hearing loss caused by mutations in OTOG when long term follow-up is present. This progression occurs mainly in the mid frequencies and mainly after 20 years of age. In one family with mutations in OTOG and one family with mutations in OTOGL additional psychophysical tests were performed. The results are similar in both families, but differ from results of normal hearing individuals. Vestibular examination showed evidence of hyporeflexia in all tested and affected family members. In addition, delayed motor development was noticed in three individuals (AII:1, CII:2 and CII:4). Novel genotype-phenotype correlations | 85 Mutations in genes encoding components of the tectorial membrane give a similar hearing loss Otogelin and otogelin-like are components of the tectorial membrane. TECTA (DFNA8/12 and DFNB21), CEACAM16 (DFNA4B) and COL11A2 (DFNA13 and DFNB53), are human deafness- associated genes that code for other proteins of the tectorial membrane 22-24. The phenotypic characteristics of these inherited types of sensorineural hearing loss are summarized in table 1. Non-syndromic hearing impairment caused by defects in one of the tectorial membrane proteins is usually characterized by a flat to U-shaped audiogram, has an early onset and is often not progressive, especially when the defects are inherited in an autosomal recessive way. These characteristics are comparable to those of the patients described here. The hearing loss is not of the cochlear conductive type despite suspected tectorial membrane involvement Psychophysical evaluation in patients with a defect in the tectorial membrane due to mutations in TECTA and COL11A2 revealed a clear cochlear conductive hearing loss. This type of hearing loss is characterized by performance in the (near-)normal range for the gap detection test, difference limen for frequency test and speech reception in noise test, elevated acoustic reflex thresholds and a parallel shift of the curve for loudness scaling 14, 15. Otogelin and otogelin-like are also components of the tectorial membrane and Simmler et al. 8 stated that the resistance of the tectorial membrane to mechanical stress produced by sound wave pressure is reduced in the absence of otogelin. Therefore, we also predicted a cochlear conductive hearing loss in patients with mutations in OTOG or OTOGL. The present psychophysical and audiological data, however, do not support this. On the other hand, the S/N value is obviously better (mean value OTOG/OTOGL patients: -1.8 dB) than that found in presbyacusis patients (+0.7 dB) (figure 6) which contradicts a sensorineural type of hearing loss. This discrepancy is possibly caused by an audibility problem: speech and noise have a broad frequency spectrum. When carrying out the speech in noise test in hearing impaired subjects, amplification should enable full audibility. This is easily acquired in case of a relatively flat hearing loss, as is found in phenotypes caused by mutations in OTOG and OTOGL. Typically, hearing loss in presbyacusis is not flat but downsloping, affecting predominantly the higher frequencies. In such patients, whenever amplification is acceptable in the low and mid-frequencies, speech sounds might still be poorly audible in the high frequencies. As shown by Killion and Christensen 25, corrections can be made to deal with this audibility problem. Following their method, the S/N ratio for the studied group of presbyacusis corrected for audibility, is approximately -1.3 dB instead of +0.7 dB. That S/N value is comparable with the mean S/N value of the patients with mutations in OTOG and DFNA13 (COLL11A2) 22, 30 Prelingual DFNB84B (OTOGL) Prelingual Prelingual 32 DFNB18B (OTOG) DFNB53 (COLL11A2) DFNB21 (TECTA) 31 Prelingual Congenital DFNA8/12 (TECTA) 28, 29 Autosomal recessive inheritance Postlingual Variable (depending on affected domain) DFNA4 (CEACAM16) 24 Autosomal dominant inheritance Onset All All All All, mid frequency dip Mid, occasionally high Mid or high, depending on affected protein domain. All Affected frequencies Mild to moderate Mild to moderate Profound Moderate to profound Mild to moderate Mild to severe Moderate Severity None Mild None None None Variable (depending on cysteine-replacing substitutions) Yes, +/- 50 dB HL Progression Table 1 Characteristics of hearing impairment caused by mutations in genes encoding components of the tectorial membrane. 86 | Chapter 2 Novel genotype-phenotype correlations | 87 OTOGL. This strengthens the suggestion that, although the defect is located in the tectorial membrane, it does not cause hearing loss of the cochlear conductive type. A possible explanation might be that the outer hair cells, with their stereocilia in contact with the tectorial membrane, do not function normally because of an ineffective connection. One should keep in mind that psychophysical tests are not easy to perform and require good concentration. Therefore, the young age of the participants might have influenced the reliability of the results of psychophysical examinations. This might explain the minor, although not significant, differences between families A and C, since the affected individuals of family A are younger than those of family C. A more accurate description can and must be made when larger numbers of (older) patients with OTOG and OTOGL mutations are available. Consequences of impaired otogelin function for hearing and balance In Otog knock-out mice, detachment of the otoconial membrane and cupulae from the neuroepithelia endorses the requirement of otogelin for anchoring the acellular membrane to the underlying neuroepithelia 8. This detachment of the otoconial membrane and cupula may explain the impaired vestibular function in the affected individuals and thereby the delayed motor development in AII:1, CII:2 en CII:4. Because of compensatory mechanisms, vestibular dysfunction will probably not have any further clinical consequences 26. Mice with a mutation in OTOG have progressive moderate to profound hearing loss. However, in histology the tectorial membrane of these mice appears to be normal. Otogelin does not seem to be involved in anchoring the tectorial membrane to the spiral limbus. However, in transmission electron microscopy some abnormal fibrillar or rod-like structures roughly parallel to the axis of the tectorial membrane were detected. Simmler et al. 7 stated that the resistance of the tectorial membrane might be reduced in the absence of otogelin, which might reduce sound transduction and leads to sound attenuation. However the results of psychophysical testing in the patients point towards a defect in the hair cells or the connection between the stereocilia and tectorial membrane. Further research is necessary to unravel a possible of role of otogelin and otogelin-like in these defects. Progression of hearing loss Progression of hearing loss is not seen nor reported in three out of four families. In family D, which carries mutations in OTOG, progression is mainly seen after the age of twenty and is relatively mild. The individuals of family C are too young to make a statement on progression in this family. The slow progression might be explained by the fact that otogelin transcription almost vanishes in the cochlea in adult mice, but otogelin labeling persists. This suggests a slow turnover process of 88 | Chapter 2 otogelin in the cochlea 27. Yariz et al. 12 also describe high levels of OTOGL transcripts in early development and down regulation in later development, which suggests involvement in the development of the structure, but a low turnover. Further follow-up or identification of older patients with hearing loss caused by mutations in OTOG or OTOGL will reveal how hearing loss presents over time. Conclusion In this paper, the audiovestibular phenotypes of two families with autosomal recessively inherited mutations in OTOG are compared with two families with mutations in OTOGL, because of the striking similarities in structure of the proteins and their localization in the tectorial membrane. So far, there are only five families known with hearing loss with underlying mutations in OTOG or OTOGL. The present results show that there are no significant phenotypic differences between all examined families. Overall one can conclude that mutations in either OTOG or OTOGL lead to a mild-to-moderate sensorineural hearing loss with a flat to gently downsloping audiogram. So far, mild progression is only seen in one family with mutations in OTOG. Clear evidence of vestibular hyporeflexia is found with relatively mild clinical consequences. Additional psychophysical examinations in two Dutch families also do not show any differences between the phenotypic expression of OTOG and OTOGL mutations. Since otogelin and otogelin-like are detected in the tectorial membrane, one could expect a cochlear conductive hearing loss, as was shown in DFNA13 and DFNA8/12 patients. However, present results of psychophysical examinations do not support this. Further research is needed to determine the exact role of otogelin and otogelin-like in the cochlea. Meanwhile, present results will improve genetic counseling of patients with mutations in OTOG or OTOGL. Novel genotype-phenotype correlations | 89 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. van Laer L, Cryns K, Smith RJ, et al. Nonsyndromic hearing loss. Ear Hear. 2003;24(4):275-88. Duman D, Tekin M. 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Twister mutant mice are defective for otogelin, a component specific to inner ear acellular membranes. Mamm Genome. 2000;11:961–6. Simmler MC, Cohen-Salmon M, El-Amraoui A, et al. Targeted disruption of Otog results in deafness and severe imbalance. Nat genet. 2000;24:139-43. Cohen-Salmon M, Mattei MG, Petit C. Mapping of the otogelin gene (OTGN) to mouse Chromosome 7 and human Chromosome 11p14.3: a candidaye for human autosomal recessive nonsyndromic deafness DFNB18. Mamm Genome. 1999;10:520-2. Schraders M, Ruiz-Palmero L, Kalay E, et al. Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment. Am J Hum Genet. 2012;91(5):883-9. Shahin H, Rahil M, Rayan AA, et al. Nonsense mutation of the stereociliar membrane protein gene PTPRQ in human hearing loss DFNB84. J Med Genet. 2010;47:643-5. Yariz KO, Duman D, Seco CZ, et al. 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Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4). Proceedings of the National Academy of Sciences of the United States of America. 2011;108(10):4218-23. 90 | Chapter 2 25. 26. 27. 28. 29. 30. 31. 32. Killion M, Christensen L. The case of the missing dots: Al and SNR loss. Hear J. 1998;51(5):32-47. Street VA, Kallman JC, Strombom PD, et al. Vestibular function in families with inherited autosomal dominant hearing loss. J Vestib Res. 2008;18(1):51-8. El-Amraoui A, Cohen-Salmon M, Petit C, et al. Spatiotemporal expression of otogelin in the developing and adult mouse inner ear. Hear Res. 2001;158:151-9. Hildebrand MS, Morin M, Meyer NC, et al. DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss. Human mutation. 2011;32(7):825-34. Plantinga RF, Brouwer APMd, Huygen PLM, et al. A novel TECTA mutation in a Dutch DFNA8/12 family confirms genotype–phenotype correlation. JARO. 2006;7:173-81. De Leenheer EMR, McGuirt WT, Kunst HPM, et al. The phenotype of DFNA13/COL11A2. Advances in oto-rhino-laryngology. 2002;61:85-91. Meyer NC, Alasti F, Nishimura CJ, et al. Identification of three novel TECTA mutations in Iranian families with autosomal recessive nonsyndromic hearing impairment at the DFNB21 locus. American journal of medical genetics Part A. 2007;143A(14):1623-9. Chen W, Kahrizi K, Meyer NC, et al. Mutation of COL11A2 causes autosomal recessive non-syndromic hearing loss at the DFNB53 locus. J Med Genet. 2005;42(10):e61. Novel genotype-phenotype correlations | 91 2.2 Nonsyndromic hearing loss caused by USH1G mutations: widening the USH1G disease spectrum A.M.M. Oonk R.A.C. van Huet J.M. Leijendeckers J. Oostrik H. Venselaar E. van Wijk A. Beynon H.P.M. Kunst C.B. Hoyng H. Kremer M. Schraders R.J.E. Pennings Ear and Hearing 2015 Mar-Apr;36(2):205-11. 94 | Chapter 2 Abstract Currently, six genes are known to be associated with Usher syndrome type I and mutations in most of these genes can also cause nonsyndromic hearing loss. The one exception is USH1G, which is currently only known to be involved in Usher syndrome type I and atypical Usher syndrome. A Dutch family with autosomal recessively inherited hearing loss was examined. Audiometric, ophthalmic and vestibular evaluations were performed besides the genetic analysis. The hearing loss had an early onset with a downsloping audiogram configuration. Slight progression of the hearing loss was seen in both affected individuals. Compound heterozygous mutations in USH1G were found to segregate with the hearing loss in this family, a missense (c.310A>G, p.(Met104Val)) and a frameshift mutation (c.780insGCAC, p.(Tyr261Alafs*96)). Extensive ophthalmic and vestibular examinations demonstrated no abnormalities that are usually associated with Usher syndrome type I. This is the first family presented with nonsyndromic hearing loss caused by mutations in USH1G. Our findings expand the phenotypic spectrum of mutations in USH1G. Novel genotype-phenotype correlations | 95 Introduction Usher syndrome is the most common cause of combined hereditary hearing and vision loss 1. It is clinically and genetically heterogeneous, but it is mainly characterized by bilateral sensorineural hearing loss and retinitis pigmentosa (RP). Vestibular dysfunction is seen in part of the cases. Based on severity of hearing loss and presence or absence of vestibular dysfunction, Davenport and Omenn distinguished three types in 1977 2. Type I is characterized by congenital, stable, severe to profound hearing loss, vestibular areflexia and RP that has an onset before puberty. Type II can be distinguished from type I based on severity of hearing loss (moderate severe), absence of vestibular dysfunction and a later onset of RP. Type III is characterized by progressive hearing loss, variable vestibular dysfunction and variable onset of RP 1, 3. Up to now, twelve loci are known for Usher syndrome and for ten of these loci the involved genes have been identified. Eight loci are known for Usher syndrome type I and the six causative genes are: MYO7A, CDH23, USH1C, PCDH15, USH1G and CIB2 4. Like in Usher syndrome, clinical and genetic heterogeneity are also seen in autosomal recessive nonsyndromic hearing loss, for which 49 genes have been identified so far 4. Interestingly, five genes known to be involved in Usher syndrome type I, MYO7A, CDH23, USH1C, PCDH15 and CIB2, are also involved in nonsyndromic hearing loss: DFNB2, DFNB12, DFNB18A, DFNB23, and DFNB48, respectively. These DFNB phenotypes are generally characterized by prelingual, severe to profound hearing loss, occasionally including minor retinal abnormalities not typical for RP 5-8. Exceptions are DFNB2, which is reported to have a more variable onset age 9, 10, and DFNB12 which can also present with moderate hearing loss, but with a progressive nature 11, 12. USH1G is located at chromosome 17q25.1 and encodes the protein SANS. Mutations in this gene have, up to now, only been described to cause Usher syndrome type I or atypical Usher syndrome 13-15. The latter is characterized as atypical since the patients did not suffer from visual problems. Ophthalmic examination, however, revealed bone spicules and atrophy of the retinal pigment epithelium (RPE), which was characterized as late-onset RP 14, 15. To our knowledge there are no reports that describe hereditary non-syndromic sensorineural hearing loss caused by mutations in USH1G. In this study, we report a Dutch family with nonsyndromic sensorineural hearing loss caused by compound heterozygous missense and frameshift mutations in USH1G. 96 | Chapter 2 Material and methods Clinical examinations A non-consanguineous Dutch family (W08-2221) was studied. The pedigree demonstrates that hearing loss is present in only one generation, which may indicate an autosomal recessive inheritance pattern (Figure 1). This study was approved by the local medical ethics committee. All participants signed an informed consent, which additionally was used to retrieve relevant data from other medical centers. All family members of the second generation completed a questionnaire on hearing and balance. Hearing impaired participants underwent complete ear, nose and throat examination including otoscopy and external ear inspection to exclude external ear deformities, previous surgery and other possible causes of hearing impairment. A computed tomography (CT) scan of the temporal bone was performed in individual II.1 in order to exclude cochlear malformations. Figure 1 Pedigree of W08-2221. Squares indicate males, circles females. Solid symbols depict affected individuals, clear symbols mean unaffected. WT means wild type. The genotypes of individuals II.2 and II.3 are not shown because of privacy reasons. Genetic analyses and molecular modeling Peripheral blood was drawn from all family members for genetic analysis. Genomic DNA was isolated from lymphocytes according to standard procedures. Single Nucleotide Polymorphism (SNP) genotypes of individuals II.1 and II.4 were determined by use of the Affymetrix® Genome-Wide Human SNP Array 6.0. All SNP array experiments were performed and data was analyzed according to the Novel genotype-phenotype correlations | 97 manufacturer’s protocol. Genotype calling and calculation of the regions of homozygosity was performed with the Affymetrix® Genotype Console Software v2.1 with use of default settings. The segregation of the genotypes for each previously reported nonsyndromic, recessive deafness gene was visually evaluated. Haplotype analysis of Variable Number Tandem Repeat (VNTR) markers was used to evaluate the family for involvement of OTOGL, PTPRQ , STRC, GJB3 and SYNE4. VNTR marker analysis was performed as described before by Schraders et al. 16 We performed Sanger sequence analysis for genes associated with autosomal recessive hearing impairment in shared genotype regions, TRIOBP, SOX10, GIPC3, MSRB3, TECTA, BSND, WHRN, TPRN, SLC26A4, SLC26A5, GRXCR1, PJVK, TMC1 and USH1G. The effect of the missense mutation on the surface and structure of the SANS protein was predicted by using a hybrid model of the protein constructed by the automatic script in YASARA 17. The structure of the area surrounding amino acid residue 104 is based on the Protein Data Bank file 1n0r, which has a sequence identity of 41% with SANS. Audiometry Pure tone audiometry was performed, according to current standards to determine hearing thresholds at frequencies of 0.25, 0.5, 1, 2, 4 and 8 kHz. To exclude conductive hearing impairment, both air and bone conduction thresholds were determined. Speech recognition scores were measured by using standard Dutch phonetically balanced consonant-vowel-consonant word lists. The maximum phoneme recognition score (mean value of both ears) was obtained from monaural performance versus intensity curves. The mean value of both ears was plotted against age and mean pure tone average (PTA) at 1, 2 and 4 kHz. These curves were compared to the curves of presbyacusis patients 18. We additionally tested acoustic reflexes, as well as loudness scaling and speech perception thresholds in individual II.1. Acoustic reflexes were measured at the contralateral ear as well as at the ipsilateral ear at 0.5, 1, 2 and 4 kHz up to the loudness discomfort level. Loudness scaling and speech perception thresholds in noise were evaluated as described before 19, 20. The results of these examinations were compared to those of normal hearing individuals, retrieved from our own clinic, and to those of presbyacusis patients 19. Prism 5.0 (Graphpad Software Inc., San Diego, CA, USA) was used to evaluate progression of hearing loss, speech recognition scores and loudness scaling by means of regression analysis. Vestibular examination Vestibular function was evaluated by electronystagmography in individual II.1. This involved calorisation and a rotary chair testing using the velocity-step test. A 98 | Chapter 2 smooth pursuit, a gaze, an optokinetic test and saccadic tests were performed to confirm normal vestibule-oculomotor function and to exclude the presence of possible central lesions. To determine the reactivity and possible vestibular preponderance of both individual vestibuli bithermal (30 and 44˚C) water irrigation of the external auditory canal was used for calorisation. For the velocity step test, the patient was seated in a rotary chair with the head ante-flexed at 30˚ to obtain a horizontal position of the horizontal semicircular canals. The chair was accelerated and when the rotatory nystagmus had subsided during constant rotation, the chair was suddenly stopped. This was done for both directions in order to calculate the possible presence of directional preponderance (asymmetry). Analyses were performed as described previously 21. Ophthalmic evaluation Retinal function was evaluated in detail in individual II.1. We performed history taking and standard ophthalmic examination including best-corrected visual acuity (BCVA), slit-lamp biomicroscopy and ophthalmoscopy. Goldmann perimetry was performed using targets V-4e, III-4e, I-4e, I-3e, I-2e and I-1e. In addition to fundus photography (Topcon TRC50IX, Topcon Corporation, Tokyo, Japan), we obtained 30˚x30˚ and 55˚x55˚ fundus autofluorescence (FAF) images (Spectralis, Heidelberg Engineering, Heidelberg, Germany). Cross-sectional images of the central and peripheral retina were obtained with a commercially available Spectral-domain optical coherence tomography (SD-OCT) instrument (Spectralis, Heidelberg Engineering, Heidelberg, Germany) using a 20°x15˚ 19-line volume scan covering the fovea as well as a 20˚ single line scan in the superior midperiphery. The patient underwent a full-field electroretinography (ERG) performed according the guidelines of the International Society for Clinical Electrophysiology of Vision (ISCEV) 22. Additionally, color vision was evaluated using Hardy-Rand-Rittler (HRR) plates. Results Clinical evaluation Family W08-2221 consists of parents and four siblings of whom two are hearing impaired: a female (II.1) and a male (II.4). Hearing loss was noted since the age of four in both siblings. Physical examination did not demonstrate any dysmorphic features suggestive of syndromic disease. A CT scan of the temporal bones in individual II.1 did not show any abnormalities. Novel genotype-phenotype correlations | 99 Genetic analyses and molecular modeling Autozygosity mapping revealed 22 homozygous regions larger than one Mb including one known gene for recessive deafness, STRC. For 17 other known autosomal recessive deafness- associated genes the genotypes segregated with the hearing loss upon manual inspection of the SNP genotypes. OTOGL, PTPRQ , STRC, GJB3 and SYNE4 were excluded by haplotype analysis of flanking VNTR markers. Mutation analysis was performed for the other genes by Sanger sequencing and three rare variants were identified. A heterozygous synonymous variant was detected in TRIOBP (c.4809T>C; p.= (p.(Asp1603Asp)), NM_001039141.2) and two variants in USH1G (NM_173477.2). One novel variant, c.780insGCAC, which causes a frameshift and a predicted premature stop codon (p.(Tyr261Alafs*96)), and a missense variant (c.310A>G; p. (Met104Val)). The USH1G variants segregated with the hearing loss in the family (Figure 1) and were not present in 350 Dutch control alleles. The insertion was also not present in the exome variant server (EVS), while the c.310G>A variant was identified in 4 out of 13002 European American and African American alleles 23. The amino acid substitution p.(Met104Val) is predicted to be benign by Polyphen2 (score 0.066; range 0-1 with 0= benign and 1= probably damaging) 24. SIFT predicts that the substitution affects protein function (score 0.03; a score ≤ 0.05 predicts the change to be damaging and >0.05 predicts it to be tolerated) 25. Mutation taster predicts the substitution to be disease causing with a probability of 0.99 (0-1) 26. Molecular modeling predicts that substitution of methionine for valine in the third ankyrin domain of SANS causes a change at the surface of this domain, and therefore possible loss of hydrophobic contacts but no change in structure of the domain (Figure 2A). For comparison, modeling of a previously described missense mutation (p.(Leu48Pro)) in patients with Usher syndrome type Ig was performed 27. This missense mutation is predicted to cause a disturbance of the protein structure (Figure 2B). Ankyrin domains are mainly formed by helical structures, and proline is known to disrupt helical structures. Therefore, it is expected that this missense mutation changes the helical structure of the protein and hence affects protein function (figure 2B). Audiometric evaluation Figure 3A demonstrates the deterioration of air conduction thresholds of individual II.1 over time. Air conduction thresholds did not differ from bone conduction thresholds. At onset, her hearing loss was moderate, but over time it progressed to severe. The audiogram configuration was initially flat to gently downsloping, but over time the curve became steeper downsloping, demonstrating significant progression especially in the high frequencies (Table 1). 100 | Chapter 2 Figure 2 A. Molecular modeling of missense variants in SANS. Close-up of the third ankyrin domain with wild type amino acid (methionine) in green and the variant (valine) in red. The change of methionine to a valine at position 104, leads to a change at the surface of the ankyrin domain. B. Close-up of the first ankyrin domain with wild type (leucine) in green and the variant (proline) in red. Insertion of a proline for a leucine at position 48 causes a change in structure, due to the interference with the helix structure of the ankyrin domain. Table 1 Annual deterioration thresholds 0.25 kHz 0.5 kHz 1 kHz 2 kHz 4 kHz 8 kHz II.1 p-value -0.25 (n.s.) 0.09 (n.s.) -0.25 (0.02) -1.51 (<0.0001) -1.71 (<0.0001) -1.60 (<0.0001) II.4 p-value -0.82 (0.03) -0.25 (n.s.) -0.09 (n.s) -0.51 (n.s.) -1.17 (0.0001) -2.02 (0.0001) Results of longitudinal regression analysis of individual binaural mean air conduction thresholds in dB/yr. Novel genotype-phenotype correlations | 101 When speech perception scores were plotted against age, scores began to deteriorate after the age of 20 years. When speech perception scores were plotted against PTA1,2,4kHz, individual II.1 clearly performed better than presbyacusis patients (Figure 4). A B dB -10 0 4.8 dB -10 0 20 10.0 20 10.3 40 15.2 40 15.2 II.1 20.9 60 II.4 4.9 20.3 60 25.6 80 80 100 100 120 120 140 140 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz Figure 3 Longitudinal binaural mean air conduction threshold data of family members II.1 (A); aged 4.8-25.6 years and II.4 (B); aged 4.9-20.3 years. Age (years) is shown with a symbol key. B 100 90 100 90 80 80 % Correct % Correct A 60 40 60 40 20 20 0 0 0 20 40 60 Age (y) 80 100 0 20 40 60 80 100 PTA 1, 2, 4 kHz Figure 4 A. Longitudinal regression analysis of mean monaural phoneme recognition score (% correct) related to age (year) of both affected family members. Scores of II.1 are indicated in grey circles, the regression line is a dashed grey curve. Scores of II.4 are indicated as black squares, the regression line is black and solid. The dashed curve indicates nonlinear regression of presbyacusis patients. B. Longitudinal regression analysis of mean monaural phoneme recognition score (% correct) related to PTA1, 2, 4 kHz (dB HL). Same legend as figure 4A. 102 | Chapter 2 Patient II.4 also showed a moderate hearing loss and progression was less outspoken over time than in case II.1. Audiogram configuration remained flat to gently downsloping (Figure 3B) but progression was significant at 0.25, 4 and 8 kHz (Table 1). Speech recognition scores plotted against age were comparable to those of individual II.1 and remained stable. However, individual II.4 had just reached the age at which the scores of individual II.1 began to deteriorate. When the scores were plotted against PTA1,2,4kHz, they were comparable to those of individual II.1. Acoustic reflexes were absent in individual II.1. Loudness scaling of II.1 showed a steeper loudness growth than one would expect from normal hearing individuals for both 0.5 kHz and 2 kHz. The signal to noise ratio was worse than the average ratio of normal hearing individuals and presbyacusis patients. Vestibular evaluation Patient II.1 complained of motion sickness. She showed a spontaneous nystagmus of 3 deg/s to the left, which was considered to be non-significant. Recording of the optokinetic nystagmus and results of all vestibule-oculomotor tests were normal. During the velocity step test, gain and start speed were slightly higher than normal; time constant and amplitude were normal. Results of calorisation were normal as well. There was a slight asymmetry between both labyrinths, to the detriment of the right vestibulum. Based on these results, vestibular function was considered normal. Ophthalmic evaluation Both patients had no visual complaints, including absence of night blindness, visual field loss and decrease in central vision. Patient II.1 presented with amblyopia of the right eye, with a suboptimal visual acuity of 20/30 after occlusion therapy, whereas visual acuity reached 20/12 in the left eye. Ophthalmic examination revealed normal anterior segments and retinas. There were no signs of RP: no bone spicules, no attenuation of the retinal vasculature, no (waxy) pallor of the optic disc or degeneration of the RPE were observed (Figure 5A). FAF images revealed normal autofluorescence of the RPE (Figure 5B) and SD-OCT revealed normal retinal architecture, including intact photoreceptor layer reflectance in the macula and midperiphery (Figure 5D and 5E). Additionally, Goldmann perimetry showed normal visual field sizes and sensibility levels and HRR plates were named correctly. Rod- and cone-derived responses on full-field ERG were normal in both eyes. We concluded that subject II.1 had normal retinal appearance and function in both eyes. The amblyopia found in this patient was not considered to be a (prodromal) symptom of an Usher syndrome phenotype. Novel genotype-phenotype correlations | 103 Figure 5 Multi-modal imaging of the right eye of patient II.1 at age 25. A. Fundus photography of central fundus of the right eye showing no abnormalities. B. Fundus autofluorescence imaging of the right eye with normal autofluorescence levels in the posterior pole. C. Infra-red en face image of the posterior pole of the right eye. The green lines indicate the location of the optical coherence tomography (OCT) images depicted in panels D and E. D. OCT image of the central retina of the right eye depicting the fovea, which reveals no retinal abnormalities and especially a normal photoreceptor layer and retinal pigment epithelium (RPE). E. OCT image of the midperipheral retina of the right eye revealing normal retinal architecture. Discussion Here, we present, to our knowledge, the first cases of non-syndromic autosomal recessively inherited hearing loss caused by mutations in USH1G. The moderate hearing loss has an onset during early childhood. Mild progression of hearing loss, especially in the higher frequencies, was observed. Resulting in a downsloping audiogram configuration. Although it was only performed in one individual, results of psychophysical examination were in line with a cochlear, sensorineural hearing loss. No vestibular abnormalities were detected. During ophthalmic examination of patient II.1, no retinal pathology was observed, especially no signs 104 | Chapter 2 of RP which is a clinical hallmark for Usher syndrome. This absence of ophthalmic abnormalities made us characterize the phenotype in individual II.1 as nonsyndromic. However, while absent at the age of 25 years, RP may develop at a later age, which is highly atypical for type I Usher syndrome. Atypical USH1G-associated Usher syndrome has been described by Kalay et al. and Bashir et al. in patients with retinal abnormalities, but without visual problems 14, 15. However, these retinal abnormalities were already observed in patients younger than individual II.1. Of the six known Usher syndrome type I genes, five were already known to be involved in nonsyndromic autosomal recessive hearing loss, except for USH1G 6, 8, 28-30 . A number of explanations have been given for the fact that mutations in these genes either lead to syndromic or nonsyndromic hearing loss. Ouyang et al. illustrated the importance of tissue-dependent alternative splicing as a cause of phenotypic heterogeneity in Usher syndrome type 1c, since harmonin has isoforms that are only expressed in the inner ear and not in the retina 5. However, to our knowledge, only one isoform is currently known for the USH1G encoded protein SANS. The difference between Usher syndrome type 1f and DFNB23 has been correlated to the mutational effect and residual function of specific protein domains. Only missense mutations that do not affect calcium binding and rigidification of protocadherin15 (PCDH15) cause DFNB23 thus far 6, 29, 31. Missense mutations are more likely to result in residual protein function than truncating mutations. Furthermore, Riazuddin et al. hypothesize that some residual function of myosin VIIA is retained in DFNB2 and that no residual function of the protein will be found in Usher syndrome type 1b 8, which was also hypothesized for CDH23 (DFNB12 and Usher syndrome type 1d) 7, 30. Families have been described with Usher syndrome type Ig carrying USH1G missense mutations that are expected to result in residual protein function. Therefore, we assume that the differences of the genotypic spectrum depend on the extent of residual protein function. In addition, other factors are probably also involved in the heterogeneity of the phenotypic spectrum of USH1G-associated disease. When compared to other USH1G genotypes, the genotype described here mostly resembles the genotype of a German family described by Weil et al. 27. In this family, one allele contains a missense mutation (c.143T>C) which leads to a substitution of proline for leucine (p.(Leu48Pro)) in the first ankyrin domain. The second mutation (c.186-187delCA) is predicted to lead to a truncated protein (p. (Ile63Leufs*71)). The phenotype in the affected members of this German family deviates from that in family W08-2221 as the former demonstrates an Usher Novel genotype-phenotype correlations | 105 syndrome type I phenotype. As shown by modeling, the change of methionine to valine at position 104 might only alter the surface of SANS, and not the overall structure (figure 2A). This may result in a reduction or loss of binding of one or more interactions partner of SANS. The p.(Leu48Pro) substitution is predicted to lead to a more severe structure alteration and predicted loss of hydrophobic contacts (Figure 2B). This may interfere more with interaction partners than the slight surface change caused by p.(Met104Val). Interacting with other proteins is essential for the function of SANS, since it is a scaffold protein and is known to bind with multiple proteins of the Usher interactome 32. Furthermore, the p. (Leu48Pro) mutation might affect binding of one or more proteins that are essential in both inner ear and retina whereas the p.(Met104Val) might affect the interaction with protein(s) that are only essential in cochlear function. To our knowledge, no interaction partners for the ankyrin domains of SANS have been identified. Questions now rise on the prevalence of nonsyndromic hearing loss based on mutations in USH1G. One is probably less vigilant on USH1G as a causal gene for nonsyndromic hearing loss since it has not been described before to be associated with a nonsyndromic type of the disease. We assume that more patients with USH1G-associated isolated hearing loss will be identified in the future, especially as the c.310A>G mutation has been found in 0.03% of alleles in the EVS database. We demonstrate that several different phenotypes can trigger one to consider USH1G as the causative gene: when a patient presents with Usher syndrome type I or with atypical Usher syndrome, but most certainly also when a patient presents with a nonsyndromic, autosomal recessive, slightly progressive hearing loss. Conclusion We describe, to our knowledge, the first family with nonsyndromic sensorineural hearing loss based on mutations in USH1G. The hearing loss has an onset during early childhood, is progressive and has a downsloping audiogram configuration. Ophthalmic and vestibular abnormalities are absent. More knowledge on interaction partners and how they bind to SANS will probably clarify the heterogeneous spectrum of phenotypes found with mutations in USH1G in the near future. 106 | Chapter 2 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Millan JM, Aller E, Jaijo T, et al. An update on the genetics of usher syndrome. 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Variable clinical features in patients with CDH23 mutations (USH1D-DFNB12). Otology & neurotology: official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology. 2004;25(5):699-706. Riazuddin S, Nazli S, Ahmed ZM, et al. Mutation spectrum of MYO7A and evaluation of a novel nonsyndromic deafness DFNB2 allele with residual function. Human mutation. 2008;29(4):502-11. Weil D, Kussel P, Blanchard S, et al. The autosomal recessive isolated deafness, DFNB2, and the Usher 1B syndrome are allelic defects of the myosin-VIIA gene. Nature genetics. 1997;16(2):191-3. Hildebrand MS, Thorne NP, Bromhead CJ, et al. Variable hearing impairment in a DFNB2 family with a novel MYO7A missense mutation. Clinical genetics. 2010;77(6):563-71. Astuto LM, Bork JM, Weston MD, et al. CDH23 mutation and phenotype heterogeneity: a profile of 107 diverse families with Usher syndrome and nonsyndromic deafness. 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Human molecular genetics. 2008;17(1):71-86. 2.3 Progressive hearing loss and vestibular dysfunction caused by a homozygous nonsense mutation in CLIC5 C.Z. Seco* A.M.M. Oonk* M. Domínguez-Ruiz J.M.T. Draaisma M. Gandía J. Oostrik K. Neveling H.P.M. Kunst L.H. Hoefsloot I. del Castillo R.J.E. Pennings H. Kremer R.J.C. Admiraal M. Schraders *These authors contributed equally Eur J Hum Genet. 2015 Feb;23(2):189-94. 110 | Chapter 2 Abstract In a consanguineous Turkish family diagnosed with autosomal recessive nonsyndromic hearing impairment (arNSHI), a homozygous region of 47.4 Mb was shared by the two affected siblings on chromosome 6p21.1-q15. This region contains 247 genes including the known deafness- associated gene MYO6. No pathogenic variants were found in MYO6 neither with sequence analysis of the coding region and splice sites nor with mRNA analysis. Subsequent candidate gene evaluation revealed CLIC5 as an excellent candidate gene. The orthologous mouse gene is mutated in the jitterbug mutant that exhibits progressive hearing impairment and vestibular dysfunction. Mutation analysis of CLIC5 revealed a homozygous nonsense mutation c.96T>A (p.(Cys32X)) that segregated with the hearing loss. Further analysis of CLIC5 in 213 arNSHI patients from mostly Dutch and Spanish origin did not reveal any additional pathogenic variants. CLIC5 mutations are thus not a common cause of arNSHI in these populations. The hearing loss in the present family had an onset in early childhood and progressed from mild to severe or even profound before the second decade. Impaired hearing is accompanied by vestibular areflexia and in one of the patients with mild renal dysfunction. Although we demonstrate that CLIC5 is expressed in many other human tissues no additional symptoms were observed in these patients. In conclusion, our results show that CLIC5 is a novel arNSHI gene involved in progressive hearing impairment, vestibular and possibly mild renal dysfunction in a family of Turkish origin. Novel genotype-phenotype correlations | 111 Introduction Hearing impairment is the most common sensory disorder worldwide and it is clinically and genetically very heterogeneous 1. Approximately 80% of early onset hereditary non-syndromic hearing impairment inherits in an autosomal recessive pattern. Currently, 80 loci and 49 genes have been identified for autosomal recessive non-syndromic hearing impairment (arNSHI), showing the great genetic heterogeneity 2. This heterogeneity might well be explained by the complexity of the auditory system. Defects in a large variety of biological processes such as gene regulation, ion homeostasis and hair bundle morphogenesis can lead to hearing impairment 3. In the last decade, homozygosity mapping using genome-wide SNP genotyping has been a powerful tool in the identification of arNSHI loci and genes 4. Lately, next generation sequencing technologies have revolutionized the genetics field and also led to the identification of novel arNSHI genes at fast pace 5. The most powerful evidence to assign a candidate gene as a novel deafness gene is to discover pathogenic variants in several families and not in controls. However, due to the large genetic heterogeneity of hearing impairment this can be very difficult even with the current technologies. This is also evident from three recently identified arNSHI genes, PNPT1, SERPINB6 and TSPEAR, for which mutations have only been described in a single family each 6-8. Further evidence to assign a candidate gene as a deafness gene can come from animal models, especially mouse models with hearing loss. Several genes essential for hearing in humans were identified after they had already been demonstrated to be associated with deafness in mice 9. Using homozygosity mapping and candidate gene analysis we identified a homozygous nonsense mutation in CLIC5 in a consanguineous Turkish family (W05-009). The orthologous mouse gene, Clic5, was described to be mutated in the jitterbug (jbg) mouse exhibiting congenital progressive hearing impairment and vestibular dysfunction due to progressive hair cell degeneration 10. Materials and Methods Subjects and clinical evaluations This study was approved by the local medical ethics committee of the Radboud university medical center and Hospital Universitario Ramon y Cajal. Signed informed consent was obtained from the parents, since all patients are minors. In addition, a signed form was used to retrieve relevant data from other medical centers. For the affected individuals of family W05-009 general physical examination was 112 | Chapter 2 performed by a pediatrician. Blood and urine samples were analyzed to evaluate renal and thyroid function. ENT examination was executed to exclude other possible causes of hearing impairment like previous ear surgery and external ear deformities. A computed tomography (CT) scan of the temporal bone was performed in order to exclude possible anatomical causes of hearing loss. Pure tone audiometry was performed according to current standards to determine hearing thresholds at 0.25, 0.5, 1, 2, 4 and 8 kHz. To exclude conductive hearing loss both air conduction and bone conduction thresholds were obtained. Classification of the hearing loss is in accordance with the GENDEAF guidelines 2. In addition, otoacoustic emissions (OAEs) were measured in individual II.3. Vestibular function was evaluated by electronystagmography and rotatory tests 11. GraphPad Prism 5.00 (GraphPad, San Diego, CA, USA) was used to perform linear regression analysis to evaluate progression of the hearing impairment. Three panels of arNSHI patients were analyzed for involvement of CLIC5. GJB2 mutations or GJB6 deletions were excluded by routine analysis in most of these patients. The first panel consisted of 76 arNSHI index patients, mostly of Dutch origin, and these were selected based on the hearing loss phenotype. They presented either with a downsloping audiogram configuration and progression of hearing loss or early onset progressive hearing loss accompanied by vestibular areflexia or hyporeflexia. The second panel consisted of 69 unrelated arNSHI sibships of Spanish origin which were not preselected based on type or severity of their hearing impairment. The third panel contained 18 arNSHI index patients of Spanish origin selected based on the hearing loss phenotype. In most of the cases, the hearing loss was postlingual (16 in childhood, at school age; two in the second decade of life) and progressive with a downsloping audiogram configuration. Homozygosity Mapping Genomic DNA was isolated from peripheral-blood lymphocytes by standard procedures. Individuals II.2 and II.3 from family W05-009 were genotyped using the Affymetrix mapping 250K NspI SNP array. All SNP array experiments were performed and analyzed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). Genotype calling and calculation of the regions of homozygosity were performed with the Genotyping Console software (Affymetrix) with the default settings. The cosegregation of the genotypes for each previously reported arNSHI gene was visually evaluated. Mutation Analysis Primers for amplification of exons and exon-intron boundaries of CLIC5 (NM_016929.4, CLIC5A and NM_001114086.1, CLIC5B), ESPN (NM_031475.2), MYO6 Novel genotype-phenotype correlations | 113 (NM_004999.3) and for mRNA analysis of MYO6 (NM_004999.3) were designed with ExonPrimer 12. Primer sequences and PCR conditions are provided in Supplemental Table S1. Amplification by PCR was performed on 40 ng of genomic DNA with Taq DNA polymerase (Roche, Mannheim, Germany). For MYO6 mRNA analysis, total RNA was isolated from Epstein-Barr-virus (EBV)-transformed lymphoblastoid cells of affected individual II.2 using the NucleoSpin RNA II kit (Machery Nagel, Düren, Germany) according to the manufacturer’s protocol. Subsequently, cDNA synthesis was performed with 1.5 µg RNA as starting material by using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s protocol. PCR reactions were performed on 2 µl cDNA with the Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). PCR fragments were purified with the use of NucleoFast 96 PCR plates (Clontech, Mountain View, CA, USA) or ExoI/FastAP (Fermentas, Vilnius, Lithuania) in accordance with the manufacturer’s protocol. Sequence analysis was performed with the ABI PRISM BigDye Terminator Cycle Sequencing V2.0 Ready Reaction kit and analyzed with the ABI PRISM 3730 DNA analyzer (Applied Biosystems Foster City, CA, USA). The presence of the CLIC5 c.96T>A transversion was investigated in 111 ethnically matched healthy controls. Exon 2 of CLIC5 was amplified and PCR products were purified as described above. Digestion of the PCR products with HpyCH4III (New England Biolabs, Ipswich, MA, USA) was performed in accordance with the manufacturer’s protocol, and restriction fragments were analyzed on gels containing 1.5% agarose and 1% low-melting agarose. The mutation removes a restriction site. Nonsense-mediated mRNA decay (NMD) evaluation EBV-transformed lymphoblastoid cell lines were established from heparin blood of individuals II.2 and II.3. Cells were grown with and without cycloheximide, a protein synthesis inhibitor, which prevents the nonsense-mediated mRNA decay process as described previously 13. Total RNA was isolated as described above. cDNA synthesis was performed with 3 µg RNA as starting material by using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s protocol. For the quantitative PCR (qPCR), specific primers (Supplemental Table S1) were designed with Primer3Plus14 and reference sequence NM_016929.4. PCRs were performed with the Applied Biosystem Fast 7900 System in accordance with the manufacturer’s protocol. The human beta glucuronidase gene (GUSB [MIM 611499]) was employed as an internal control. PCR mixtures were prepared with the Power Syber Green Master Mix (Applied Biosystems) in accordance with the manufacturer’s protocol. Temperatures and reaction times for PCR were as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C. All reactions were performed in duplicate. Relative gene expression 114 | Chapter 2 levels were determined with the delta delta Ct method as described previously 15. CLIC5 Expression Profile RNA derived from adult heart, retina, brain and kidney was purchased from Clontech (Mountain View, CA, USA). RNA derived from adult skeletal muscle, liver, duodenum, testis, spleen, thymus, and placenta were purchased from Stratagene (La Jolla, CA, USA) and bone marrow from Bio-chain (Newark, CA, USA). RNA derived from fetal brain, colon, kidney, stomach, spleen, heart, skeletal muscle, lung and thymus was purchased from Stratagene. In addition, RNA was isolated from adult lung, fetal cochlea and fetal liver as described previously 16. The inner ear was derived from a fetus at 8 weeks of gestation and the other fetal tissues from fetuses at 20 – 21 weeks of gestation. cDNA synthesis, primer design (Supplemental Table S1) and qPCR analysis were performed as described above. The forward primer was located on the boundary of exons 2 and 3, and the reverse primer in exon 3. This enabled detection of all CLIC5 isoforms affected by the nonsense mutation that is located in exon 2. Relative gene expression levels were determined with the comparative delta Ct method as described previously 17. Results Hearing loss and vestibular dysfunction in family W05-009 ENT examination and CT scanning did not reveal an apparent cause of hearing impairment in the two affected children of family W05-009. The parents have normal hearing and are first cousins of Turkish ancestry (Figure 1A). The patients presented with an early onset sensorineural hearing loss since the bone conduction thresholds did not differ from the air conduction thresholds. The hearing impairment probably was not congenital since individual II.2 passed a behavioural reflex audiometry test which is performed between 7 and 9 months of age (Ewing test) and individual II.3 had normal hearing at the age of 3 months during brainstem evoked response audiometry. The hearing loss started mildly, affecting the mid and high frequencies mostly. OAEs were not present in individual II.3 at the age of 4 months. It progressed to a severe-to-profound hearing loss with a gently to steeply downsloping audiogram configuration as is shown in Figure 1B. Longitudinal linear regression analysis indicated significant progression in all frequencies (Figure 1C). Individual II.2 received a cochlear implant at the age of 11 years. Five years post-implantation, the speech recognition scores were 88% (using the standard Dutch phonetically balanced consonant-vocal-consonant word lists) 18 . Initial motor milestones were reported to be normal, but later in life balance problems did occur e.g. difficulties with walking in the dark and cycling. Vestibular Novel genotype-phenotype correlations | 115 areflexia was found in both individuals during the rotatory test at the age of 16 years (II.2) and 11 years (II.3). General physical examination showed that both children had a normal height and weight. No other abnormalities besides the hearing impairment were noted in individual II.2. On history, there were no signs of any other abnormalities in individual II.3. Thyroid function was also normal in individuals II.2 and II.3. Renal function was normal in II.2. However, in individual II.3 a blood pressure of 127/73 to 129/88 mmHg (90% value for this age and length: 120/76 mmHg) was measured and an elevated albumin/creatinine ratio was detected at the last two visits in the outpatient clinic (9.2 and 3.8 mg/mmol, respectively, normal value <2.5 mg/mmol) during urine analysis at the age of 11 years. The glomerular filtration rate, calculated with the Schwartz equation, was normal with a value of 114 ml/ min/1.73 m2 (normal > 90 ml/min/1.73 m2). Since the elevated blood pressure and albumin/creatinine ratio were seen in repeated measurements, these might be the first signs of a nephropathy. However, no other more invasive studies were performed, since the renal function was overall only mildly affected. Subsequent renal function follow-up of patient II.3 in the future will be needed to determine whether it is indeed a nephropathy. Homozygosity mapping confines a critical region on chromosome 6p21.1-q15 To identify genes for arNSHI, homozygosity mapping was performed in some large series of familial and isolated arNSHI patients living in the Netherlands. In family W05-009 homozygosity mapping revealed a homozygous region of 47.4 Mb on chromosome 6p21.1-q15 (Figure 2A). This was the largest homozygous region shared by the two affected individuals. This region contains the known deafnessassociated gene MYO6. There were 15 other shared homozygous regions larger than 1 Mb (Supplemental Table S2), and in one of them ESPN is located, which is another gene known for arNSHI. Mutation analysis was performed for all exons and exon-intron boundaries of ESPN and MYO6, which revealed no putative causative variants. Since MYO6 was located in the largest shared homozygous region, MYO6 mRNA was analyzed by RT-PCR. No PCR-products of an aberrant size were indentified and also sequence analysis of the PCR products did not reveal indications for aberrant splicing. To exclude compound heterozygous or allozygous mutations in the other known arNSHI genes, cosegregation of the genotypes was visually evaluated for each gene. PNPT1, ILDR1, RDX, TECTA, OTOGL, PTPRQ and OTOA were present within the genotype shared regions. OTOA could be excluded by short tandem repeat (STR) marker analysis in the family. No putative pathogenic variants were identified in the other genes by sequence analysis of the exons and exon-intron boundaries. 116 | Chapter 2 A nonsense mutation in CLIC5 causes arNSHI Since no pathogenic variants could be identified in MYO6 or ESPN we hypothesized that another gene would be underlying the hearing loss in this family. Therefore, we continued with the evaluation of the 247 annotated genes in the largest homozygous region, 6p21.1-q15 19. For three of the genes, mutations in the orthologous mouse gene cause deafness. Slc17a5 (MGI:1924105)20 and Bmp5 (MGI:88181) are associated with mixed and conductive hearing loss in the mouse, respectively. The third gene, Clic5, was described to underlie progressive A I.2 p.Cys32Ter WT I.1 p.Cys32Ter WT II.1 WT WT II.3 p.Cys32Ter p.Cys32Ter II.2 p.Cys32Ter p.Cys32Ter B dB -10 0 20 dB II.2 4.6 y 3.1 y 6.4 y 20 4.4 y 8.2 y 40 16.0 y p95 60 5.2 y 40 6.5 y 60 80 80 100 100 120 120 140 II.3 -10 0 8.1 y 11.2 y p95 140 .25 .5 1 2 4 8 kHz .25 .5 1 2 4 8 kHz Figure 1 A. Pedigree of family W05-009 and segregation of the CLIC5 c.96T>A variant. B. Longitudinal binaural mean air-conduction pure tone thresholds are shown of affected members of family W05-009. Age (years) is shown with a symbol key. The p95 line, matched for age and sex, indicates that 95% of the population has thresholds lower than these. C. Regression analysis of longitudinal binaural mean air conduction threshold data for each frequency separately. Circles indicate individual II.2, squares indicate individual II.3. Annual threshold deterioration is shown behind the symbol key for each frequency. 10 Figure 1 Continued. 0 5 10 5 10 15 20 0 5 10 15 20 -100 -100 -5.3 dB HL/y -2.6 dB HL/y 0 -100 0 4 kHz 20 -50 0 15 -50 -4.0 dB HL/y -2.5 dB HL/y 20 -50 0 2 kHz 15 -100 -100 -6.0 dB HL/y -5.7 dB HL/y 0 -100 5 0.5 kHz -50 -5.9 dB HL/y -4.1 dB HL/y 0 -50 0 0.25 kHz -50 0 0 0 5 5 Age (y) 10 8 kHz 10 1 kHz 20 15 20 -4.8 dB HL/y (n.s.) -2.4 dB HL/y 15 -3.9 dB HL/y -1.4 dB HL/y Novel genotype-phenotype correlations C | 117 db HL 118 | Chapter 2 sensorineural hearing loss and vestibular dysfunction in the jbg mouse mutant 10. Therefore, CLIC5 represented an excellent candidate gene. Mutation analysis of CLIC5 revealed a homozygous nonsense variant c.96T>A (p.(Cys32X)) (Figure 2B), which segregated with the hearing impairment in family W05-009 (Figure 1A). This variant was not present in 222 Turkish control alleles, the Exome Variant Server21 and the Nijmegen in-house exome database (1302 exomes). This variant was submitted to the Leiden Open Variant Database 22. The homozygous CLIC5 c.96C>T mutation is predicted to result in NMD, since it creates a premature stop codon (p.(Cys32X)) more than 54 bp upstream of the 3’-most intron 23. However, we could not confirm NMD. The relative CLIC5 mRNA expression is comparable in cycloheximide treated and untreated patient EBV-cell lines (157.24% vs. 157.89%, respectively) and higher - albeit not significantly - than in controls (set at 100%) as shown in Supplemental figure 1. A B Figure 2 A. Schematic overview of the chromosomal region 6q21.1-q15 showing the homozygous regions (black bars) identified in the affected individuals of family W05-009. The homozygous region of individual II.2 delimits the region to 47.4 Mb. Mb positions and chromosome bands are according to the UCSC genome browser (GRCh37). B. Partial sequences are shown of CLIC5 exon 2 from an affected member, a parent and an unaffected sib of family W05-009. The predicted amino acid changes and the surrounding amino acids are indicated above the sequence. Mutated nucleotides are marked by an arrowhead. As reference sequence NM_016929.4 was employed. Novel genotype-phenotype correlations | 119 For identification of other families with CLIC5 mutations, sequence analysis of the coding region and splice sites of CLIC5 was performed in 76 arNSHI index patients, mostly of Dutch origin, and 18 Spanish arNSHI index patients. These patients were preselected based on the hearing loss and vestibular phenotype as described in the materials and methods section. No putatively causative variants were identified in these patients. In a parallel approach, compatibility with linkage to DFNB102 was investigated in a panel of 69 unrelated arNSHI sibships of Spanish origin. These were genotyped for STR-markers D6S459, D6S1920, D6S1632 and D6S1638, which flank CLIC5. Haplotype analysis revealed compatibility with linkage of the disease locus to these markers in 18 sibships. Sequence analysis of CLIC5 in the index cases of these sibships did not reveal any putative pathogenic variants. Finally, data of 50 whole exome sequence analysis of arNSHI patients, mainly of Dutch origin were evaluated for the presence of variants in CLIC5. These patients were not preselected based on the type or severity of the hearing loss. No putatively pathogenic variants were identified in this cohort. CLIC5 is expressed in the human inner ear The expression of CLIC5 was studied via qPCR in human fetal inner ear and compared to that in additional 13 adult and 10 fetal-stage human tissues (Figure 3). Since this was performed in two separate experiments for adult and fetal tissues, fetal inner ear was included in both for comparison. The tissues were not derived from fetuses of the same gestational stage; therefore, a direct comparison of the transcript levels cannot be made. In fetal tissues, CLIC5 transcript levels were highest in skeletal muscle, kidney, spleen, heart and colon. A lower expression level was found in fetal brain, thymus, lung and stomach. Expression in the fetal inner ear was 26 fold higher than in fetal liver in which the expression level was the lowest. In adult tissues, the highest expression levels were found in heart, lung, skeletal muscle and kidney and there was a gradual decrease through retina, spleen, brain, placenta, duodenum and thymus. Finally, the lowest expression levels were found in bone marrow, testis and liver. We can conclude that CLIC5 is widely expressed in both fetal and adult human tissues. 120 | Chapter 2 Relative CLIC5 expression in fetal tissues A 10000 1000 100 10 B Sk r ve h om Li ac ng St Lu ai n Th ym us Br rt Co lo n In ne re ar H ea n Sp le e el et al m us cl e Ki dn ey 1 Relative CLIC5 expression in adult tissues 10000 1000 100 10 r r li nn er ea ve Li ta Fe cl e Ki dn ey Re ti na Sp le en Br ai n Pl ac en D ta uo de nu m T Bo hy m ne us m ar ro w Te st is m us ng Sk el et al Lu H ea rt 1 Figure 3 CLIC5 expression profile in human tissues. Relative CLIC5 mRNA levels as determined by qPCR in human fetal (A) and adult (B) tissues. The relative expression values were determined by using the comparative delta Ct method 17. The expression levels are relative to those in liver, which showed the lowest CLIC5 expression of all the tissues that were tested. Discussion We present a homozygous nonsense mutation, c.96T>A (p.(Cys32X)) in CLIC5 (DFNB102) as a cause of sensorineural hearing impairment accompanied by vestibular dysfunction. This nonsense variant will lead to an early truncation of the protein. Screening a series of arNSHI index patients did not reveal additional causative variants, suggesting that mutations in CLIC5 are not a common cause of arNSHI neither in the Netherlands nor in Spain. Initially, the hearing loss in the affected individuals of family W05-009 was mild, mainly affecting the mid and high frequencies, and then it progressed to severe to profound. They also showed vestibular areflexia in the second decade of life. The combination of progression to profound hearing loss and complete vestibular dysfunction in the patients of family W05-009 resembles the phenotype of the jbg mutants. Auditory-evoked brainstem responses (ABRs) at 1-5 months of age in jbg Novel genotype-phenotype correlations | 121 mutant mice, which are null for Clic5, were 40-50 dB above those of wildtype mice. By 7 months of age their hearing loss had progressed to complete deafness due to progressive hair bundle degeneration and a reduced density of spiral ganglion cells 10. The vestibular hair cells of jbg mice also showed a progressive degeneration. In the crista ampullaris, the number of vestibular hair cells was reduced at 3 months and hair cells were nearly absent at 12 months of age 10. Besides inner ear dysfunction, the phenotype in humans and mice with Clic5 mutations may well overlap with respect to the renal phenotype. The jbg mice have abnormalities in the foot processes of the kidney podocytes leading to proteinuria 24,25. The elevated albumin/creatinine ratio and pre-hypertension in affected individual II.3 indicate mild renal dysfunction and may well be the first signs of a nephropathy. Therefore, follow-up of renal function is indicated for individual II.3 but also for his hearing impaired sister. In addition to the inner ear and kidney phenotypes, the jbg mutant mice also exhibit emphysema-like lung pathology, hyperactivity and resistance to dietinduced obesity 26. Characteristics of lung emphysema and hyperactivity were excluded in family W05-009 by history taking. The affected individuals had normal height and weight. No other gross abnormalities in the organs of the jbg mutants were detected and no other complaints from the affected siblings were reported so far. The lack of symptoms in organs with relatively high CLIC5 expression (Figure 3) points towards redundancy for CLIC5 function there. CLIC5 belongs to a family of chloride intracellular channel proteins, but its protein structure differs from other typical ion channel proteins. CLICs (CLIC1-6) show no sequence homology to any known channel family, but significant homology to glutathione S-transferases in the core region 27. Moreover, some of the CLIC proteins, including CLIC5, are not only integral membrane proteins, but are also found as soluble proteins in the cytoplasm 28. In the inner ear, CLIC5 localizes to stereocilia of both the cochlear and vestibular hair cells and on the surface of Kolliker’s organ during cochlea development in mice 10. Specifically, CLIC5 is predominantly present at the base of stereocilia and, to lesser extent, in the cell bodies of hair cells in the region surrounding the cuticular plate 10. CLIC5 was initially identified in placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin and ezrin 29. Although the function of CLIC5 is still elusive, there are several facts that support its role in stereocilia integrity. Firstly, radixin immunostaining is reduced in the hair bundle of jbg mice where it colocalizes with Clic5 10. This suggests that Clic5 is needed for proper radixin activity, so when interacting with the ERM (erzin, radixin and moesin) complex, Clic5 may stabilize connections between the plasma membrane and the filamentous actin core 10. Secondly, Clic5 functions as an 122 | Chapter 2 adapter between the plasma membrane of podocytes and the actin cytoskeleton by facilitating the interaction between ezrin and podocalyxin 24, 25, 30. Thirdly, a recent study proposes that Clic5 functions as part of a multiprotein linker complex in companion with radixin, erzin and taperin 31. Protein tyrosine phosphatase receptor Q (Ptprq), which is mislocalized as radixin in the jbg mice, and Myosin VI, key regulator of the proper localization of Ptprq32, might well participate in this complex too. Radixin33, Ptprq32, 34 Myosin VI35 and Clic5 deficient mice10 show loss of stereocilia at the bundle vertex and fusion of stereocilia in postnatal stage. This suggests that this multiprotein complex is essential for stable membrane-cytoskeletal attachments at the stereocilia base. Conclusion We show that a homozygous nonsense mutation in CLIC5 (p.(Cys32X)) underlies the progressive hearing impairment, vestibular and possibly mild renal dysfunction in a family of Turkish origin. CLIC5 mutations do not seem to be a common cause of arNSHI in the Dutch and Spanish populations. Further screening of CLIC5 in other populations will provide important information about the frequency of CLIC5 mutations and may aid to further define the phenotype associated with CLIC5 mutations. 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The Journal of cell biology. 2004;166(4):559-70. Goodyear RJ, Legan PK, Wright MB, et al. A receptor-like inositol lipid phosphatase is required for the maturation of developing cochlear hair bundles. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2003;23(27):9208-19. Self T, Sobe T, Copeland NG, et al. Role of myosin VI in the differentiation of cochlear hair cells. Developmental biology. 1999;214(2):331-41. Novel genotype-phenotype correlations | 125 Supplemental Table S1 Primer sequences and PCR conditions for genomic PCR, sequence analysis and QPCR. Target Oligonucleotides Product Annealing size (bps) Temperature (°C) Primers for genomic PCR and sequence analysis of CLIC5, MYO6 and ESPN CLIC5 Exon 1 Forward primer CCAGGACCAGCTCTGTCTC Reverse primer AACCTGCCTTCCTCCATTC CLIC5 Exon 2 Forward primer TTATTGAGCCCTTACATGCTG Reverse primer ACTTGCCTTTGCAGACTCTC CLIC5 Exon 3 Forward primer ACAGGCATGTTGGATAGCAC Reverse primer GTCACTGGGTTACCCTTTCC CLIC5 Exon 4 Forward primer GCTGGAAAGGGAAGTATTGC Reverse primer CTCTGGCCCAAGTATTTGAC CLIC5 Exon 5 Forward primer TCCAGGTATTCTGCAGTGTG Reverse primer AGGTTCTTGACCAACAGGTG CLIC5 Exon 6 Forward primer AAAGAAGACAGCACTGCCAC Reverse primer GCCTCAAGGCAGTGATACAG CLIC5 Exon 1a* MYO6 Exon 1 MYO6 Exon 2 MYO6 Exon 3 MYO6 Exon 4 MYO6 Exon 5 MYO6 Exon 6 MYO6 Exon 7 MYO6 Exon 8 MYO6 Exon 9 Forward primer TCCAATGGACTTGTGGTTTG Reverse primer TGACTGATTTAGTCCCTCCG Forward primer TCCGTAGCCGTGACGTG Reverse primer TCGGGCAGTGAACAGAAG Forward primer TGGGCAGATGTGTTTGTTAG Reverse primer TGCTTTCCCAAATATCTACCTC Forward primer GTATGCAACCAATTAAGCCC Reverse primer TAGATTGTTTCTGAACCCGC Forward primer TAGGGTTTACATCAGCCAGG Reverse primer TCAAGCATGATTTTCTGTTAGAC Forward primer TGGGTCCCTATAAAGATCAGG Reverse primer CCCTGTGTAAAATACTTGCTCC Forward primer TGATTTCTTTAAGAGTAAGTGGTCC Reverse primer TACAGTGAGCTGTGGTGGTG Forward primer TGATGATCTAGGTTTCAGTTTTATATG Reverse primer TGAGAGAAGAACATTCCAGACC Forward primer GCATGAGCCACTGTGTCC Reverse primer TGCAAATACAGCACCAACTG Forward primer AACCTCTTTGATAGACAAATGG Reverse primer CAGGCTCAAGCAATCCATC 733 56 484 56 653 56 306 56 431 56 427 56 885 56 532 56 388 56 262 56 337 56 471 56 322 58 238 56 324 56 651 58 126 | Chapter 2 Supplemental Table S1 Continued. Target Oligonucleotides Product Annealing size (bps) Temperature (°C) Primers for genomic PCR and sequence analysis of CLIC5, MYO6 and ESPN MYO6 Exon 10 MYO6 Exon 11 MYO6 Exon 12 MYO6 Exon 13 MYO6 Exon 14 MYO6 Exon 15 MYO6 Exon 16 MYO6 Exon 17 MYO6 Exon 18 MYO6 Exon 19 MYO6 Exon 20 Forward primer TTCATGGTTGGCACTATTTG Reverse primer CAACAAGAGGTATAAAGTATTACACTG Forward primer AGTGCATTAATTGACCTGGTG Reverse primer TTTAAGCAAACTGCATATAAAGAGAC Forward primer AAAAGTTACAAATAAGCCTTGCC Reverse primer TTTTGATCTACCAAGCTCAGG Forward primer GTTTAGGTGCACTCTGTGGC Reverse primer CATATTATGAAATGTGAGTGTGGAC Forward primer TGAGAACATTTGGGAAGTATAGAAC Reverse primer ATACACCATCCATCCACACC Forward primer TTCAGAAACAGTGCAAAATTC Reverse primer CTTCCAGGCAGTAATACAGAAG Forward primer TGTTGTTTCTGATCAGTCCTTG Reverse primer TCCATCTAAGGAAGATACTGTGC Forward primer CTTACACCTATTTTCTTTCCTAAGAG Reverse primer TCACCACTAGTCAGTGTGGAC Forward primer AAGTTCCTTTGGACAGAGCC Reverse primer CAAATATCAACAATGCAGGG Forward primer CCAGGAGGTTGTTAAACTGG Reverse primer CAGGGTTACTATGCCTACTTGC Forward primer CCCAGCCTTGAGTTCTTTAG Reverse primer AAGATGAAAATTTTATTGCACTG MYO6 Exon 21-22 Forward primer Reverse primer MYO6 Exon 23 MYO6 Exon 24 MYO6 Exon 25 MYO6 Exon 26 MYO6 Exon 27 TCTCATAAATTGCCCGTTTC 353 56 363 58 375 56 376 56 351 56 295 56 389 56 500 56 390 56 267 56 361 56 580 56 354 56 307 56 375 56 460 56 309 56 TGTTGTTAGTGACCATATAATTCAAG Forward primer TGCCAAGGCCTATGTAATTG Reverse primer AGCATCACCTCTGAGACAGC Forward primer GGTCGTCAAGATTTCATGTTTC Reverse primer AAAACCTGAGTATCCAAACTGC Forward primer TTGAAAACAGAAGTGAAATACCC Reverse primer GTCTCAACACATTTTGTAATTTTG Forward primer GCTGTATTTGCATATTGGAGTAG Reverse primer TTGTCATTAACCACTGTCAATACC Forward primer TTCCCAATCTGTTACCTTTG Reverse primer TTGATCTCCTGACCTCGTG Novel genotype-phenotype correlations | 127 Supplemental Table S1 Continued. Target Oligonucleotides Product Annealing size (bps) Temperature (°C) Primers for genomic PCR and sequence analysis of CLIC5, MYO6 and ESPN MYO6 Exon 28 MYO6 Exon 29 MYO6 Exon 30 MYO6 Exon 31 MYO6 Exon 32 MYO6 Exon 33 MYO6 Exon 34 MYO6 Exon 35_1 MYO6 Exon 35_2 MYO6 Exon 35_3 ESPN Exon 1 ESPN Exon 2 ESPN Exon 3-5 Forward primer GGGGCAGTTATGCTTTCC Reverse primer TTCTGCATGGAAATGAGAGG Forward primer CACAAATTTGCACAATCCAG Reverse primer AGCACCATACAAGAGCATTAAAC Forward primer TGTGTTACGGCTAGATTTGTTG Reverse primer TCATGTAACAGGTTCTGGTCC Forward primer TCCGGTTTTCAAACTTATGC Reverse primer GTGCATTCATGGACCAAAAG Forward primer GCTTATCCTTATGAATAATTAGCTTAC Reverse primer GCCATCAAGGCTGTATTAGG Forward primer ACTTTTCAGTCACCACCTCG Reverse primer TCCACTGAAAATTGTAGCAAAAC Forward primer GGGGTATATTTTAGGATTAAAGGC Reverse primer TGGAAATGTGATTTAACCGC Forward primer ATTGAAAGGGTCCTTGATGG Reverse primer TGCCTTGATCATTTTAAGTGG Forward primer CCGCTGTAATTCCCAAAAC Reverse primer TCCAGTTAAGCCACTATGCC Forward primer GCACAATGTGTGTTGCTGTC Reverse primer CCTAACTGAGGTAATCTTTCTAGGG Forward primer ATTCGAACCCAGTTTTGCTG Reverse primer CCACCCACTTCCAGGACTAC Forward primer AGGAAGGGTGGAGAGATC Reverse primer ATGTTGAGTGGGAGCCATTT Forward primer GAGGTCAGACACAGCAGGTG Reverse primer AGCGTGGGTTTCCAGTTATG Forward primer AGGTGAGCTGCACCGACGTG Reverse primer TGACCTCTAGCTCCCCGTTC ESPN Exon 6 Forward primer GGAACCTGGGTCCTGCTG Reverse primer CCTCCCCATGTTTAAGAGCA ESPN Exon 7 Forward primer TGGTCTTCCCCCAGTAAGTG Reverse primer TACTCTCCTCCCAGTCCAGTG ESPN Exon 3-5S# ESPN Exon 8 Forward primer GCTGCCCACTGTGAGAACC Reverse primer GGGAGGCCCTTTCTAGCTG 336 56 271 56 309 56 367 56 431 56 296 56 438 58 831 56 614 56 658 56 1010 58 859 58 1954 58 NA NA 552 58 673 58 933 58 128 | Chapter 2 Supplemental Table S1 Continued. Target Oligonucleotides Product Annealing size (bps) Temperature (°C) Primers for genomic PCR and sequence analysis of CLIC5, MYO6 and ESPN ESPN Exon 9 ESPN Exon 10 ESPN Exon 11-12 ESPN Exon 13 Forward primer CCATCCAATCTTGGCTTAGG Reverse primer ACTGGTGACAGTGCAGGTGA Forward primer CACAGTGTTCTCAGGCATCG Reverse primer GATGGGCTGTGCATCCAG Forward primer TCCCAGCTACTTGTTCTCTGC Reverse primer GTGGGGTTAGGTTAACTGAAGG Forward primer CTCAGTAACCCACCCCTTG Reverse primer ATCTGGGTCAGAGAGGAAGG 377 58 444 58 691 58 741 58 826 56 787 56 738 56 851 56 750 56 754 56 115 60 80 60 Primers for MYO6 mRNA analysis MYO6 mRNA Ex1-9 MYO6 mRNA Ex8-13 MYO6 mRNA Ex13-19 MYO6 mRNA Ex18-26 MYO6 mRNA Ex25-31 MYO6 mRNA Ex30/31-35 Forward primer TTCACCCGTACAGGTAGCC Reverse primer TTCCTCTTTGCCTTGAACAC Forward primer CTTTGGAAATGCGAAGACTG Reverse primer TCCAATAAAATAGGATGATGTTTC Forward primer GCAAGCAAACAATGCTCG Reverse primer CTTCGAAGTTTATCCAGAAGC Forward primer TGAATCCAGAGATAAGTTTATACGG Reverse primer GTTCCTGCGTCATCATAGTG Forward primer TGATGGTCTGGTTAAGGTGG Reverse primer TCACGTAGTTCTGCATATTTCC Forward primer TTTGAGTAGAGGTCCTGCTG Reverse primer GGGCACACACCCTACCTAAG Forward primer CGGCAACTGTCCTTTCTCTC Reverse primer GGCTAGGTTGTGCAGGTCAG Primer for QPCR CLIC5 Exon2-3 GUSB Exon2-3 Forward primer AGAGTGGTGCTGAGGATTGG Reverse primer CCCTCATGCTCTAGCGTGTC The reference sequences for exon numbering CLIC5, MYO6 and ESPN were NM_016929.4, NM_004999.3 and NM_031475.2, respectively. *This is the first exon of an alternative CLIC5 transcript (NM_001114086.1) # These primers were only used for sequence analysis. rs2477828 rs196004 rs10915317 rs11152920 rs17238567 rs1912515 rs8105809 rs1447595 rs2159579 rs10503951 rs1124531 rs9302387 rs308409 rs16840977 rs1631550 rs7118543 SNP_A-4193053 SNP_A-2227424 SNP_A-1794682 SNP_A-2090773 SNP_A-2152234 SNP_A-2013197 SNP_A-2303650 SNP_A-2194601 SNP_A-4212479 SNP_A-1993002 SNP_A-2146287 SNP_A-2206226 SNP_A-4227577 SNP_A-4195575 SNP_A-4203661 SNP_A-1826210 SNP_A-2013513 SNP_A-2048940 SNP_A-4239672 SNP_A-2214677 SNP_A-2138415 SNP_A-1860565 SNP_A-4216078 SNP_A-2215144 SNP_A-1974496 SNP_A-2059039 SNP_A-4224094 SNP_A-1980966 SNP_A-2155415 SNP_A-2070006 SNP_A-2061244 SNP_A-2248124 Name End SNP rs4483549 rs2979684 rs277638 rs3113377 rs11642765 rs891974 rs6996639 rs7800245 rs10513030 rs3745836 rs11018855 rs1366419 rs783403 rs12119878 rs153219 rs2057276 rsId 11 8 3 4 16 5 8 7 3 19 11 5 7 1 16 6 Chr. 89949097 23786381 99034331 123752692 20478724 169773687 33665680 84845546 135476532 55123464 88287451 20964216 48809712 5081066 23966086 41430495 Start position 90955972 24817441 100079447 124834671 21582734 170902780 34843712 86103733 136877493 56652592 89835267 22916852 50903934 9218721 29215298 88865821 End position Genomic position were determined using the UCSC Genome Browser website (http://genome-euro.ucsc.edu/index.html, hg19). rsId Name Start SNP 1.01 1.03 1.05 1.08 1.10 1.13 1.18 1.26 1.40 1.53 1.55 1.95 2.09 4.16 5.25 47.38 Size (Mb) Supplemental Table S2 Overview of homozygous regions shared by the affected individuals of family W05-009. - - - - - - - - - - - - - ESPN - MYO6 Known arNSHI gene Novel genotype-phenotype correlations | 129 200 150 100 50 0 mRNA expression CLIC5 (% of normal) 130 | Chapter 2 Controle (-CH) -CH +CH Supplemental Figure 1 The homozygous mutation CLIC5 c.96T>A (p.(Cys32Ter)) in family W05-009 does not result in nonsense mediated mRNA decay (NMD). Shown is the mean expression of CLIC5 in Epstein-Barr-virus (EBV)-transformed lymphoblastoid cells of affected individuals II.2 and II.3 (n=2) and controls (n=10) treated with (+CH) and without (-CH) cycloheximide. Quantifications were normalized against GUSB expression. Differences in expression between the affected individuals and 10 controls were calculated by using the delta delta Ct method.11 The p-value was derived from the standard score (Z-value) calculated for untreated samples (-CH) as compared to the normal distribution of the 10 controls. Since we assume a lower expression level as a result of NMD, a one-sided test was enough to reject the null hypothesis (p=0.376). Also, no difference was seen in CLIC5 expression between treated and untreated patient samples. In conclusion, occurrence of NMD could not be confirmed. Novel genotype-phenotype correlations | 131 3 Expanding a genotypephenotype correlation 3.1 Vestibular function and temporal bone imaging in DFNB1 A.M.M. Oonk A.J. Beynon T.A. Peters H.P.M. Kunst R.J.C. Admiraal H. Kremer B. Verbist R.J.E. Pennings Hearing Research 2015 July (Epub ahead of print) 136 | Chapter 3 Abstract DFNB1 is the most prevalent type of hereditary hearing impairment known nowadays and the audiometric phenotype is very heterogeneous. There is, however, no consensus in literature on vestibular and imaging characteristics. Vestibular function and imaging results of 44 DFNB1 patients were evaluated in this retrospective study. All patients displayed a response during rotational velocity step testing. In 65% of the cases, the caloric results were within normal range bilaterally. The video head impulse test was normal in all patients. In 34.4% of the CT scans one or more temporal bone anomalies were found. The various anomalies found, were present in small numbers and none seemed convincingly linked to a specific DFNB1genotype. The group of DFNB1 patients presented here is the largest thus far evaluated for their vestibular function. From this study, it can be assumed that DFNB1 is not associated with vestibular dysfunction or specific temporal bone anomalies. Expanding a genotype-phenotype correlation | 137 Introduction DFNB1 is the most common type of autosomal recessive hearing impairment and is caused by mutations in GJB2 and/or a deletion of GJB6. Up to 40% of the autosomal recessively inherited sensorineural hearing impairments is caused by defects in these two genes 1. Many different pathogenic mutations in GJB2 have been identified to cause hearing impairment. Because of the high incidence of mutations in GJB2 and deletions of GJB6 in different populations, it is recommended to first screen for DFNB1 in patients with suspected autosomal recessive hearing loss or in isolated cases with a congenital or prelingual hearing impairment 2, 3. The audiological phenotype of DFNB1 is highly variable. The onset is most often prelingual or congenital. In most cases, hearing impairment is stable, however, when it is not congenital it can also be progressive. The severity of hearing impairment varies between mild to profound. Audiogram configuration can vary from flat to downsloping, which therefore is not pathognomonic 1. Snoekcx et al. 4 performed a large multicenter study to correlate the different GJB2 genotypes to the audiometric phenotype. They concluded that the degree of hearing impairment associated with biallelic truncating mutations was significantly more severe than hearing impairment associated with biallelic non-truncating mutations. A genotype-phenotype correlation does not only comprise audiometric features, but also vestibular and imaging characteristics. The available information in literature on vestibular function is based on small numbers of patients and both imaging and vestibular results are not consistent 5-9 . Several studies have evaluated vestibular function in DFNB1 patients. Denoyelle et al. and Cohn et al. described a genotype- phenotype correlation study for DFNB1 patients 5-6. Both have retrospectively evaluated vestibular function based on vestibular testing. Cohn et al. 5 described a normal vestibular function in 11 out of 13 patients. The deviating results of the two other patients were assigned to immaturity of the vestibular system in a premature birth at 31 weeks gestation and to recurrent vestibulopathy related to migrainous vertigo. Denoyelle et al. 6 reported on 10 DFNB1 patients with normal vestibular function. Dodson et al. 10 assessed subjective vestibular function in DFNB1 patients by means of a survey study. Patients with DFNB1 had significantly more vestibular problems than other hearing impaired individuals. On the other hand, there were no subjective vestibular problems in DFNB1 patients studied by Kasai et al. 9. In this study six out of seven patients demonstrated unilateral abnormal responses during vestibular examination. In the study by Todt et al. 7 only two out of seven patients had normal vestibular responses. They, therefore, conclude that mutations in GJB2 138 | Chapter 3 are not only responsible for hearing impairment but also for vestibular dysfunction. The studies by Denoyelle et al. 6 and Cohn et al. 5 also evaluate temporal bone imaging in 19 and 23 patients, respectively. All 42 computed tomography (CT) scans did not display temporal bone anomalies. Temporal bone anatomy was also assessed by Propst et al. 8 by means of CT scans in 53 DFNB1 patients. In over 50% of their cases temporal bone anomalies were found. Among the most prevalent anomalies are a dilated endolymphatic fossa and an enlarged vestibular aqueduct. These anomalies can be associated with vestibular problems. Considering the varieties in findings in the different studies we evaluated both vestibular test results and imaging results in 44 DFNB1 patients from our clinic. This study further defines the genotype-phenotype correlation in DFNB1 patients. Patients and methods Patients Patients diagnosed with DFNB1 in our clinic in the time period 2002-2014 were eligible for inclusion in this retrospective study (n=65). DFNB1 patients were included when vestibular results or imaging results were available. Since vestibular function assessment and imaging are standard items of the cochlear implant selection procedure, 37 patients were cochlear implant users. The diagnosis of DFNB1 was based on two pathogenic mutations in GJB2, two pathogenic deletions in GJB6 or a pathogenic mutation in GJB2 in combination with a pathogenic deletion in GJB6. Hearing impaired offspring of parents both diagnosed with DFNB1 were also considered to have DFNB1. General features and genetic characteristics were collected by means of a chart review. Vestibular testing The vestibular function was evaluated in 28 patients. To confirm normal vestibularoculomotor function and to exclude possible central lesions smooth pursuit, gaze, optokinetic, and saccadic tests were performed. Vestibular function was evaluated by electronystagmography. This involved rotary chair testing using the velocity-step test (VST) and caloric irrigation. For the VST, the patient was seated in a rotary chair. To obtain a horizontal position of the horizontal semicircular canals the head was ante-flexed at 30 degrees. To provoke a nystagmus, the chair is accelerated with 2˚/s2 to 0.25 Hertz. After a plateau time of 60 seconds, the chair was suddenly stopped with a deceleration of 200˚/s2, inducing a physiological response, i.e. a nystagmus in opposite direction. The maximum slow phase velocity of the nystagmus and time constant were recorded. This was done for both directions to calculate the possible presence of the Expanding a genotype-phenotype correlation | 139 nystagmus’ directional preponderance (asymmetry). The reactivity and possible vestibular preponderance of individual vestibules were determined by caloric testing using bithermal (30 and 44°C) water irrigation of the external auditory canal (n=21). Analyses were performed as described previously 11. In 10 patients, additionally a video Head Impulse Test (vHIT) has been performed to evaluate the individual function of the semicircular canals in the higher frequency domain. The patient has to focus on a dot on the wall while the examiner makes a short but rapid movement with the head in the plane of the canal. All three canals are examined individually. The eye movements were recorded by a camera (vHIT Ulmer, Synapsys, Marseille, France) 12. In 17 patients vestibular function was evaluated after cochlear implantation. Three patients were too young to complete the VST and/ or caloric test reliably because of fatigue or loss of concentration. In three patients the tests was considered to be unreliable due to ear abnormalities (otitis media, tympanic membrane perforation). In four patients the results were not available for evaluation. In seven patients the VST was not performed according to the procedure described here, therefore results were considered unreliable. These patients were excluded from vestibular function analysis. Temporal bone imaging Temporal bone imaging by means of computed tomography (CT) or magnetic resonance imaging (MRI) has been evaluated in 41 patients. A CT scan was available for 33 patients but was evaluated in 32 patients. One CT scan was excluded from evaluation since a combined approach tympanotomy altered the anatomy. The temporal bones of three of these patients were also evaluated by MRI scan. In nine patients only an MRI scan of the temporal bone was performed. All scans were displayed to an experienced head-, neck- and neuroradiologist (BV) and an experienced otologist (RP) using IMPAX (version 6.5.3.1005 AGFA healthcare Mortsel, Belgium). Both ears were evaluated separately according to a structured list of items (size and aspect of external auditory canal, middle ear, ossicles, mastoid, cochlea, vestibule, semicircular canals (SCC), vascular structures, endolymphatic fossa/vestibular aqueduct) . Precise measurements of the cochlear height, width, length size of the bony island were executed with the electronic calipers available in IMPAX. All CT scans were systematically scored on 33 items 13. The MRI scans were scored on 14 items. 140 | Chapter 3 Results General characteristics Forty-four patients were included in this study. Forty-three patients were diagnosed with DFNB1 based on two mutations in GJB2 and/or GJB6. An additional patient was included because both parents were diagnosed with DFNB1 (table 1). Twenty-two female and 22 male patients were evaluated. The mean age at vestibular testing was 14.4 years (range 0.38-68.4) and the mean age during temporal bone imaging was 9.6 years (range 0.21-62.3). In 25 patients results of both VST and temporal bone imaging were available. In sixteen patients only imaging results were available, in three patients only vestibular results were present. In general, these patients were cochlear implant candidates and had profound hearing loss with a congenital onset. Two patients had a moderate to severe hearing impairment. Temporal bone imaging abnormalities were not seen in these patients and vestibular testing was not performed. In four patients, history was not taken on vestibular complaints or motor milestone development issues. Five patients complained about balance problems or demonstrated delayed motor milestones. Two of them displayed normal results on the VST and one showed hyporeflexia on VST and areflexia during calorisation. Temporal bone imaging demonstrated an opacification of the mastoid and middle ear in two patients (age 0.5-1.1 years old) and another patient with delayed motor milestones demonstrated a superior semicircular canal dehiscence. Vestibular tests Eye movements were evaluated in 20 patients. In two patients oculomotor test responses were deviant. One patient presented with slow responses during the saccadic and optokinetic test. However, VST, calorics and vHIT displayed normal responses in this patient. Another patient demonstrated a disturbed optokinetic response to the right. VST revealed a vestibular hypofunction and an areflexia of the right vestibulum during calorics. This was tested before cochlear implantation. The results of the vestibular tests are displayed in figure 1. Twenty-one patients had normal VST responses, of which 12 also showed normal calorics results. Six patients demonstrated hyporeflexia during VST (aged 3.6- 42.2), four of them were tested after cochlear implantation. The two who were tested before implantation displayed unilateral areflexia during calorics (tested at 45.7 and 51.5 years). One of these two patients demonstrated normal vHIT results. Homozygous c.35delG: 2 del GJB6/ IVS1+1G>A: 1 Homozygous c.35delG: 7 del GJB6/ c.35delG: 1 c.35delG/ c.71G>A:1 c.35delG/ IVS1+1G>A:1 c.35delG/ c.139G>T: 1 c.313-326del14 del GJB6: 1 c.407dup/ c.71G>A: 1 Normal n=1 Post implantation n=1 n=1 Not applied Homozygous del GJB6: 1 c.35delG/ c.229T>C: 1 c.-23+1G>A/ c.238C>A: 1 Normal n=1 Post implantation n=3 Normal bilateral n=3 Hyporefle f xia n=6 Homozygous c.35delG: 1 del GJB6/ c.35delG: 1 Normal n=1 Pre implantation n=2 Arefle f xia unilateral n=2 Homozygous c.23+1G>A: 1 Not applied n=1 Post implantation n=1 Hyperrefle f xia bilateral n=1 Hyperrefle f xia n=1 Homozygous c.35delG: 9 del GJB6/ c.35delG: 1 IVS1+1G>A/ del GJB6: 1 c.35delG/ c.427C>T: 1 c.35delG/ c.229T>C: 2 homozygous c.235delG: 1 c.101T>C/ c.283G>A: 1 Not applied n=16 Figure 1 Flowchart of vestibular and genetic results. The figure starts at the top with the total of patients evaluated in this study. When the figure is tracked down the results of the VST, calorisation test and vHit are presented, respectively. At the bottom the different genotypes for these specific results are presented. VST: velocity step test, vHIT: video head impulse test, del: deletion. Homozygous c.35delG: 3 del GJB6/ c.35delG: 2 parents DFNB1: 1 Mutations Post implantation n=3 Normal n=1 Post implantation n=1 Hyporefle f xia unilateral n=3 Normal n=5 Post implantation n=8 n=6 n=21 Normal n=1 Normal bilateral n=12 Not applied Calorics vHIT n=10 Normal n=21 VST N=28 n=44 Expanding a genotype-phenotype correlation | 141 142 | Chapter 3 Table 1 Overview of mutations and/ or deletions of GJB2/ GJB6 in the study population. Mutation 1 Mutation 2 N (%) c.35delG (p.(G12fs)) c.35delG (p.(G12fs)) 22 (50.0) c.35delG (p.(G12fs)) deletion GJB6 5 (11.4) c.35delG (p.(G12fs)) c.229T>C (p.(W77R)) 3 (6.8) c.35delG (p.(G12fs)) c.IVS1+1G>A 1 (2.3) c.35delG (p.(G12fs)) c.71G>A (p.(W24X)) 1 (2.3) c.35delG (p.(G12fs)) c.427C>T (p.(R143W)) 1 (2.3) c.35delG (p.(G12fs)) c.139G>T (p.(E47X)) 1 (2.3) c.35delG (p.(G12fs)) c.313-326del14 1 (2.3) deletion GJB6 deletion GJB6 1 (2.3) deletion GJB6 c.313-326del14 1 (2.3) deletion GJB6 c.IVS1+1G>A 1 (2.3) c.-23+1G>A c.-23+1G>A 1 (2.3) c.235delG (p.(L79fs)) c.235delG (p.(L79fs)) 1 (2.3) c.407dup (p.(Y136X)) c.71G>A (p.(W24X)) 1 (2.3) c.238C>A (p.(Q80K)) c.-23+1G>A 1 (2.3) c.283G>A (p.(V95M)) c.101T>C (p.(M34T)) Both parents deaf due to DFNB1 1 (2.3) 1 (2.3) Between brackets the protein change is noted. Imaging results Table 2 summarizes the results of the evaluation of temporal bone imaging. In 34,4% of the CT scans a temporal bone anomaly was detected (i.e. enlarged endolymphatic fossa, semicircular canal dehiscence, bifide malleus, hyper- or hypoplasia of the cochlea/ semicircular canal). Evaluation of the CT scans demonstrated a (partially) opacified middle ear in seven patients. The age of these patients ranged from 0.21-1.10 years and the opacification was considered to be based on otitis media with effusion. Evaluation of the labyrinth demonstrated minor abnormalities. The measured cochlear height was within normal range in 28 patients (4.4 mm-5.9 mm; <4.4 mm is categorized as hypoplasia, >5.9 mm is defined as hyperplasia) 13. The median measured cochlear height was 5.3 mm. In two patients the cochlear height was unilaterally 4.1 and 4.3 mm (hypoplasia), respectively. Another patient displayed a hyperplasia of the cochlea unilaterally (6.0 mm)(Figure 2 and 3). In the remaining patient the cochlear height could not be assessed due to absent coronal section. Expanding a genotype-phenotype correlation | 143 Figure 2 Different sizes of the cochlea. A. Normal size. B. Hypoplasia. C. Hyperplasia. The CT scan of fifteen patients demonstrated opacification in different degrees of the horizontal semicircular canal (SCC). In 10 patients this was present bilaterally. The size of the bony island of the horizontal SCC is correlated to vestibular abnormalities. In one patient the bony island of the left labyrinth measured 5.2 mm (>4.8 mm is defined as hyperplasia). The size in the remaining 63 ears ranged from 2.9 mm – 4.8 mm (normal 2.6 mm - 4.8 mm) 13 (Figure 3 and 4). The median size of the bony island was 3.9 mm. The superior SCC appeared dehiscent in three patients (unilaterally), clinically there were no indications for a SSCC dehiscence. In four patients an enlarged endolymphatic fossa was present, two cases showed this bilaterally (Figure 5). The endolymphatic fossa was not visible in seven patients and therefore not assessable. Twelve MRI scans of in total 24 ears did not show any abnormalities except for one patient with a subarachnoidal cyst as an incidental finding. 144 | Chapter 3 6 Size (mm) 5 4 la is ny Bo Co ch l ea rh ei gh nd t 3 Figure 3 Scatter plot of the cochlear height and bony island size. Line indicates the median. The filled symbols are the deviating results, hypoplasia or hyperplasia. The upper and lower limit of the normal range is indicated by dashed lines. Figure 4 Different appearances of the horizontal semicircular canal. A. Normal appearance (white arrow). B. Opacification of the horizontal semicircular canal (white arrow). C. Enlarged bony island (white arrow). Expanding a genotype-phenotype correlation | 145 Figure 5 Variations of the endolymphatic fossa. A. Normal appearance (white arrow). B. Enlarged endolymphatic fossa (white arrow). Correlation between vestibular and radiological findings The more prevalent radiological findings in this study are combined with vestibular results in figure 6. The patient with unilateral cochlear hypoplasia had normal results on VST and calorics. The patient with hyperplasia of the cochlea also displayed normal VST results, results of calorics were not available. The patient with hyperplasia of the horizontal SCC presented with a normal VST and calorics response. Truncating versus non-truncating mutations Thirty-nine patients presented with homozygous truncating mutations. Four patients presented with compound heterozygous truncating/ non-truncating mutations. VST results were available in two out of these four patients and both showed hyporeflexia, although caloric results were normal and both patients were tested after cochlear implantation. In three of these four patients temporal bone imaging results were present, none demonstrated temporal bone anomalies. One patient presented with heterozygous non-truncating mutations, temporal bone imaging did not display any anomalies. Jugular bulb Size 50 None 1 (1/0) Shape 14 (12/1) Present Short 28 Dehiscence SSCC 3 (3/0) Bifid malleus 1 (1/0) Emissary vein Hypoplastic Aspect 61 Normal 6 12 4 3 (3/0) Lateralized Sigmoid sinus HSCC 25 (5/10) 16 (2/7) 12 (2/5) 1 (1/0) Opacification na Different temporal bone characteristics indicated per ear. Numbers between brackets indicate number of patients with the characteristics unilateral/bilateral. *possibility of >1 anomaly in one ear. EAC: external auditory canal; SCC: semi circular canal (H: horizontal; S: superior); EF: endolymphatic fossa; VA: vestibular aqueduct; Na: not assessed. 17 (11/3) 30 VA 6 (2/2) 1 (1/0) 47 46 EF High riding 38 SCC* 1 (1/0) 4 (0/2) Enlarged Normal 58 Cochlea Vascular variants 63 48 Mastoid 52 Middle ear Ossicles 59 Normal EAC Ears (n=52) Table 2 Temporal bone imaging characteristics. 146 | Chapter 3 Normal n=1 vHIT Arefle f xia uni n=1 Normal n=2 Normal n=1 Calorisation vHIT Normal n=1 Normal n=1 Normal n=1 Hyporefle f xia n=1 homozygous c.35delG: 3 c.35delG/ c.del GJB6: 1 c.35delG/ c.139G>T: 1 c.-23+1G>A/c.238C>A: 1 Normal n=1 hyporefle f xia uni n=1 Normal n=4 Not assessable EF n=6 c.35delG/c.71G>A :1 c.IVS1+1G>A/c.delGJB6: 1 homozygous c.35delG: 9 c.35delG/ c.del GJB6: 2 c.35delG/ c.427C>T: 1 parents DFNB1: 1 Hyporefle f xia uni n=1 Figure 6 Flowchart of common radiological findings on computed tomography scan combined with vestibular and genetic results. The figure starts at the top with the temporal bone anomaly and when the figure is followed down the results of the VST, calorisation and vHit (when performed), respectively, are presented. At the bottom the different genotypes from the patients with that specific temporal bone anomaly are presented. HSCC: horizontal semicircular canal; SSCC: superior semicircular canal, EF: endolymphatic fossa, VST: velocity step test, uni: unilateral, vHIT: video Head Impulse Test, del: deletion. homozygous c.35delG: 2 c.35delG/ del GJB6: 1 parents DFNB1: 1 Normal n=1 Hyporefle f xia n=1 Enlarged EF n=4 Homozygous c.35delG: 2 c.IVS1+1G>A/ c.del GJB6: 1 Normal n=2 Mutations Normal n=3 Normal n=3 Normal n=9 Normal n=1 Hyporefle f xia n=1 Opacificatio acif n HSCC n=15 SSCC dehiscence n=3 VST Temporal bone anomaly Mutations Normal n=1 Calorisation VST Temporal bone anomaly Expanding a genotype-phenotype correlation | 147 148 | Chapter 3 Discussion Because of the relatively high incidence of DFNB1 the audiological phenotype has been extensively studied previously. The audiological phenotype is very divergent. Vestibular function and imaging of the temporal bones remain underexposed in these descriptions. In addition, the scarce reports on vestibular function and imaging in DFNB1 patients are not consistent with each other. This retrospective study presents an evaluation of vestibular function and imaging results, including CT as well as MRI in a relatively large number of DFNB1 patients (n= 44). Based on the present vestibular examinations and temporal bone imaging, our results in general show that neither vestibular dysfunction nor evident temporal bone anomalies are associated with DFNB1. Radiological phenotype of DFNB1 In 34,4% (11/32) of the CT scans one or more asymptomatic temporal bone anomalies were identified. This is a higher percentage than previously reported for DFNB1 patients (0-11%) 5, 14-19. However, much less than the percentage reported by Propst et al. 8 (72%). The temporal bone anomalies found in the previously mentioned studies are very diverse. Each anomaly is only detected in a limited number of cases, except for the anomalies mentioned by Propst et al. (table 3). They found an enlarged endolymphatic fossa (28.3%), a modiolus deficiency (24.7%) and an EVA (14.2%) in a substantial proportion of their cases. In 10,3% of the temporal bones in which the endolymphatic fossa could be assessed, it was enlarged in our patients. A modiolus deficiency or EVA were not identified in any of our cases. The number of DFNB1 patients with an enlarged endolymphatic fossa did not significantly differ from age-matched normal hearing controls in the study of Propst et al. Given the fact that the enlargement is only found in a limited number of patients, where it could not be linked to a specific GJB2/ GJB6 genotype, we draw the conclusion that DFNB1 is not associated with an increased prevalence of an enlarged endolymphatic fossa. However, it cannot be completely ruled out that specific GJB2 mutations present with an increased risk for an enlarged vestibular aqueduct. Opacification of the horizontal semicircular canal is a remarkable detail, present in 15 patients. Since the scans were of optimal quality and were evaluated with utmost care but the opacification could not be linked to vestibular dysfunction, we consider this as a true finding without clinical significance rather than an artifact. All together, a variety of abnormalities were found in this group of patients, each in small numbers and none seemed consistently linked to a specific genotype. 113 21 35 53 Preciado et al. 14 Lee et al. 17 Propst et al. 8 38 2 1 12 0 2 1 3 Number of patients with temporal bone anomalies 72.0% 5.7% 4.8% 10.6% 0% 8.3% 5.6% 11% Percentage of patients with temporal bone anomalies EVA; enlarged vestibular aqueduct. SCC: semicircular canal. CNC: cochlear nerve canal. EF: endolymphatic fossa, HSCC: horizontal semicircular canal. SSCC: superior semicircular canal. VA: vestibular aqueduct. 19 Kenna et al. 19 24 Cohn et al. 5 Angeli et al. 18 Kochhar et al. 16 15 27 Lipan et al. 18 Number of patients Dilated EF (28.3%) Modiolus deficiency (24.7%) EVA (14.2%) Hypoplastic HSCC (7.5%) Hypoplastic cochlea (3.8%) Dilated ampulla (3.8%) Dehiscent HSCC (3.8%) Dehiscent SSCC (1.9%) Pericochlear lucency (0.9%) VA extending in ampulla (0.9%) EVA Borderline EVA SCC abnormalities Internal auditory meati asymmetry Modiolus deficiency and CNC hypoplasia Bilateral CNC hypoplasia EVA Mondini malformation EVA Mondini malformation Internal auditory canal with bulbous shape Dilated vestibule and HSCC Type of anomaly Table 3 Summary of studies reporting on temporal bone imaging in DFNB1 patients. Expanding a genotype-phenotype correlation | 149 150 | Chapter 3 No vestibular abnormalities in DFNB1 patients A vestibular response was found in all patients during the VST, additionally all patients had at least one excitable vestibule during calorics and, as far as data was available, the vHIT was normal in all patients. In two patients the vHIT was normal, while calorics demonstrated areflexia or hyporeflexia, respectively. This difference can be explained by the fact that the vHIT stimulates the vestibule in the 4-7 Hz frequency range, while caloric irrigation stimulates the vestibule in a much lower frequency range i.e. 0.003 Hz 20. There was no correlation between vestibular function and the underlying genotype (homozygous truncating mutations or compound heterozygous non-truncating/ truncating mutations). This conclusion is, however, based on a small number of patients. The vestibular function is considered to be normal in DFNB1 patients, however since this is a retrospective study we should take into account that the vestibular function was evaluated after cochlear implantation in 17 patients. On the other hand, this only endorses the point of normal vestibular function in DFNB1 patients. With a mean age of 14.4 at vestibular testing and 9.6 at temporal bone imaging, a relatively young group of patients is presented here. Since the auditory phenotype of DFNB1 with a congenital onset in general is not progressive, the vestibular phenotype is not expected to deter later in life. However, further research should include older patients since vestibular dysfunction could appear later in life together with temporal bone imaging abnormalities. This is for example seen in patients with DFNA9, a dominantly inherited progressive cochleovestibular disorder. DFNA9 patients develop focal sclerosis of the semicircular canals and cochlea later in life 21. Evaluation of connexin function and dysfunction in the vestibule GJB2 and GJB6 encode connexin 26 and connexin 30, respectively. Co-localized connexin 26 and connexin 30 form intercellular gap junctions which, in addition to others, are responsible for potassium diffusion in the membranous labyrinth. This is necessary in maintaining cochlear ion homeostasis 22. When one of both genes is mutated, insufficient or less functional gap junctions will be formed. This will change the potassium concentration in the endolymph, which is toxic to hair cells 23. Connexin 26 and connexin 30 are expressed in the sensory and non-sensory epithelia of the saccule, utricle and ampullae of mice. A crucial difference in these structures is the presence of dark cells in the utricle and ampullae 24. These dark cells are also involved in potassium recirculation by other potassium regulating channels: NaK-ATPase, NaKCl and KCNQ1/KCNE1 25, 26 . In the absence of sufficient connexin 26 and connexin 30 based gap junctions, these dark cells may correct the ratio of potassium in the endolymph in the utricle and ampullae. Hence, a change in gap junction may mainly affect the function of the saccule. Expanding a genotype-phenotype correlation | 151 The function of the utricle and saccule can be evaluated by measuring vestibular evoked myogenic potentials (VEMPs). Cervical VEMPs have been tested in DFNB1 patients in two previous studies by Todt et al. 7 and Kasai et al. 9. They performed VEMPs in nine patients with biallelic GJB2 mutations. Two patients did not show any response, three patients only demonstrated a unilateral response and four patients responded normal. When the genetic defect would affect saccular function as suggested above, it would be logically to find this deterioration bilaterally, regarding the symmetry of the hearing impairment. Although the number of patients in the studies by Todt et al. 7 and Kasai et al. 9 are relatively small, the results indicate no or minimal effect of the genetic defect on the saccule. With the VST, caloric test and the vHIT, the focus in this retrospective study is on the semicircular canals, and not on the utricle and saccule. To rule out or confirm a functional role of connexin 26 or connexin 30 in the saccule, we recommend to examine cervical VEMPs in future DFNB1 patients. In connexin 30 knockout mice, the early development of hair cells in the saccule was not affected. The hair cells, however, degenerate similar to hair cells of the cochlea. When connexin 26 is overexpressed in these mice, the morphology of the saccular hair cells was restored. The hair cells of the utricle and ampullae were not affected in knockout mice 24. Insufficient quantity of gap junctions probably leads to saccular hair cell death. This raises the question which combination of mutations and how severe these mutations need to be, before the gap junction quantity is poor enough for saccular dysfunction. And what will the clinical consequences be, or will this be compensated well? Conclusion In this study, a large variety of temporal bone anomalies in DFNB1 patients were identified. These anomalies were only found in small numbers and not linked to specific genotypes. Therefore, we assume that DFNB1 is not associated with specific temporal bone anomalies. In addition, no significant vestibular dysfunction is seen in DFNB1 patients, nor was there a relation between vestibular findings and imaging results. This retrospective study did not evaluate saccular function. Because of a hypothetical relationship between saccular dysfunction and DFNB1 it is recommended that future studies evaluate this shortcoming, in order to assess saccular function in DFNB1 patients. Since temporal bone anomalies and vestibular dysfunction (except potential saccular dysfunction) could not be linked to DFNB1, it is currently not recommended to routinely perform temporal bone imaging or vestibular testing in DFNB1 patients. From a scientific point of view these diagnostic tools should, however, be considered 152 | Chapter 3 in order to expand the genotype-phenotype correlation or to emphasize the current correlation. During the cochlear implant selection procedure or when clinical features of vestibular dysfunction or temporal bone anomalies (e.g. mixed hearing loss) are present, additional vestibular testing or temporal bone imaging should be considered. Before implantation of a second cochlear implant, vestibular function should always be re-assessed in order to rule out vestibular areflexia of the first implanted ear. Expanding a genotype-phenotype correlation | 153 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. del Castillo FJ, del Castillo I. The DFNB1 subtype of autosomal recessive non-syndromic hearing impairment. Frontiers in bioscience. 2011;16:3252-74. Ardle BM, Bitner-Glindzicz M. Investigation of the child with permanent hearing impairment. Archives of disease in childhood Education and practice edition. 2010;95(1):14-23. Pandya A, Arnos KS, Xia XJ, et al. Frequency and distribution of GJB2 (connexin 26) and GJB6 (connexin 30) mutations in a large North American repository of deaf probands. Genetics in medicine : official journal of the American College of Medical Genetics. 2003;5(4):295-303. Snoeckx RL, Huygen PL, Feldmann D, et al. GJB2 mutations and degree of hearing loss: a multicenter study. American journal of human genetics. 2005;77(6):945-57. Cohn ES, Kelley PM, Fowler TW, et al. Clinical studies of families with hearing loss attributable to mutations in the connexin 26 gene (GJB2/DFNB1). Pediatrics. 1999;103(3):546-50. Denoyelle F, Marlin S, Weil D, et al. Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: implications for genetic counselling. Lancet. 1999;353(9161): 1298-303. Todt I, Hennies HC, Basta D, et al. Vestibular dysfunction of patients with mutations of Connexin 26. Neuroreport. 2005;16(11):1179-81. Propst EJ, Blaser S, Stockley TL, et al. Temporal bone imaging in GJB2 deafness. The Laryngoscope. 2006;116(12):2178-86. Kasai M, Hayashi C, Iizuka T, et al. Vestibular function of patients with profound deafness related to GJB2 mutation. Acta oto-laryngologica. 2010;130(9):990-5. Dodson KM, Blanton SH, Welch KO, et al. Vestibular dysfunction in DFNB1 deafness. American journal of medical genetics Part A. 2011;155A(5):993-1000. Theunissen EJ, Huygen PL, Folgering HT. Vestibular hyperreactivity and hyperventilation. Clinical otolaryngology and allied sciences. 1986;11(3):161-9. Macdougall HG, McGarvie LA, Halmagyi GM, et al. The video Head Impulse Test (vHIT) detects vertical semicircular canal dysfunction. PloS one. 2013;8(4):e61488. Purcell DD, Fischbein NJ, Patel A, et al. Two temporal bone computed tomography measurements increase recognition of malformations and predict sensorineural hearing loss. The Laryngoscope. 2006;116(8):1439-46. Preciado DA, Lim LH, Cohen AP, et al. A diagnostic paradigm for childhood idiopathic sensorineural hearing loss. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery. 2004;131(6):804-9. Angeli SI. Phenotype/genotype correlations in a DFNB1 cohort with ethnical diversity. The Laryngoscope. 2008;118(11):2014-23. Kochhar A, Angeli SI, Dave SP, et al. Imaging correlation of children with DFNB1 vs non-DFNB1 hearing loss. 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Functional significance of channels and transporters expressed in the inner ear and kidney. American journal of physiology Cell physiology. 2007;293(4):C1187-208. Expanding a genotype-phenotype correlation | 155 4 Psychosocial impact of a genetic diagnosis 4.1 Psychological impact of a genetic diagnosis on hearing impairment an exploratory study A.M.M. Oonk S. Ariens H.P.M. Kunst R.J.C. Admiraal H. Kremer R.J.E. Pennings Submitted 160 | Chapter 4 Abstract Genetic testing for hereditary hearing impairment becomes more routinely available as a diagnostic tool in the outpatient clinic. Little is, however, known on the psychological impact of a genetic diagnosis. In order to evaluate this impact, an exploratory study was conducted. Prospectively, 48 individuals who underwent genetic testing for hereditary hearing impairment were included in this study. They were asked to fill out the following different questionnaires: Hospital Anxiety Depression Scale, Impact of Event Scale, Self Efficacy 24, Illness Cognition Questionnaire and the Inventory for Social Reliance. The questionnaires were filled out at three time points; before genetic testing, directly after counseling on the either positive or negative test result and six weeks thereafter. No statistical differences were found between the groups who received a genetic diagnosis for their hearing impairment (positive group) and the group who did not (negative group). Special attention to the psychological well being should be offered to hearing impaired patients who seek a genetic diagnosis for their hearing impairment. In addition, the psychological impact of sensorineural hearing impairment might be larger than the impact of a genetic diagnosis itself. Based on the current exploratory study, there are no psychological reasons in favor or against genetic testing for hereditary hearing impairment. In the future, studies should focus on the impact of the genetic diagnosis retrieved from whole exome sequencing and on the impact of a syndromic or nonsyndromic hearing impairment. Psychosocial impact of a genetic diagnosis | 161 Introduction Early onset hearing impairment has a hereditary cause in about 50% of cases 1. Nowadays, over 75 genes have been identified to be associated with hereditary hearing impairment 2. In addition, genetic testing for a hereditary cause of hearing impairment is increasingly available in out-patient clinics as routine diagnostics. After establishing a genetic diagnosis for hearing impairment, the individual and his/her relatives need proper counseling. This counseling should focus on inheritance, prognosis of hearing impairment, potential vestibular dysfunction, additional symptoms 3 and possible effects of rehabilitation 4. So far, only little is known on the psychological impact of a genetic diagnosis for hearing impairment 5. More research has been performed on the impact of genetic testing in, for example, Huntington disease or hereditary breast and ovarian cancer. Some patients with these disorders demonstrated an increase in psychological distress after receiving a positive test result. The observed psychological distress mostly vanished within a short period of time 6, 7. On the other hand, patients who received negative results displayed a decrease in psychological distress 8. Results of counseling on a genetic diagnosis in these more frequently studied diseases can probably not be transposed to hereditary hearing impairment because they may be fatal or may require medical intervention to avert this fatality. In addition, research on the impact of genetic testing for Huntington disease or hereditary breast and ovarian cancer also concerns pre-symptomatic testing. Genetic testing for hereditary hearing impairment in adults is usually performed when hearing impairment is already symptomatic. Humans naturally seek explanations to elucidate the cause of an event. Some of these causes are controllable (e.g. will power), and some are uncontrollable (e.g. genetics). Research by Gordon et al. demonstrated that patients who were counseled on a neutral genotype (no mutations with beneficial or adverse effects) had an increase in health perception and self concepts, which was designated as increased psychological well being. This was dedicated to a probable increase in personal control 9. Based on the study of Gordon et al., Palmer et al. hypothesized that hearing impaired individuals who receive a genetic diagnosis for their hearing impairment would display an increase in psychological well being compared to those without a genetic diagnosis 5. They tested this hypothesis in adults with an early onset hearing impairment, who were tested for mutations in GJB2/ GJB6 (DFNB1). A positive test result led to an increase in perceived personal control and a decrease in anxiety level. Patients who received a negative result, had an increase in anxiety 162 | Chapter 4 level and decrease in perceived personal control. These effects were still substantially present six months after diagnosis and were more outspoken and lasted longer than to be expected from previous research 5. Palmer et al. focused only on patients who were tested on mutations in GJB2/ GJB6 (DFNB1). The hearing impairment in these patients is mostly congenital and severe to profound. The present exploratory study aimed to evaluate the psychological impact of a genetic diagnosis on nonsyndromic hearing impairment in adult individuals in order to improve counseling of genetic hearing impairment. Patients and methods Patients This study was approved by the local medical ethics committee. Individuals were requested to participate in this study when they fulfilled three criteria. First, subjects had to be at least 18 years of age. Second, a hereditary cause of their hearing impairment was suspected. Third, a single gene mutation analysis was applied in order to identify the cause of their hearing impairment. Individuals who were not mentally competent and/ or not able to read and understand Dutch were excluded from the study. Subjects were recruited over a 15 month period of time (September 2013- November 2014). The study protocol contained three time points of evaluation. The first time point concerned inclusion, the moment genetic testing was requested (T0). The second evaluation concerned the period from counseling on the result of the genetic test (either positive or negative) until two weeks after (T1). The third time point was six weeks after the questionnaires of T1 were completed (T2). At the time of these three evaluation time points, individuals were asked to complete several questionnaires. The questionnaires of T0 were completed at the outpatient clinic. The questionnaires of T1 and T2 were sent and returned by mail. Questionnaires Demographic data Demographic data were collected at T0 by means of a questionnaire. This self-constructed questionnaire assessed educational level 10, (history of) psychological complaints, medication, offspring or the desire to have children, hereditary disease in the family and reasons for genetic testing. This questionnaire also assessed hearing characteristics including onset, progression and rehabilitation of hearing impairment. Psychosocial impact of a genetic diagnosis | 163 Illness Cognition Questionnaire (ICQ ) The ICQ was developed to evaluate three subscales: acceptation, helplessness and disease benefits of a chronic disease 11. In this study the ICQ was focused on hearing impairment as chronic disease. The questionnaire was completed at T0. The questionnaire contains 18 items, six for each subscale. Each item can be scored according to a four points Likert scale. Scores range from six to twenty-four for each subscale. A higher score indicates a higher level of acceptation, helplessness or disease benefits. The crohnbach т ranged from 0.84-0.90 for the three subscales 11. Inventory for Social Reliance (ISR) The ISR is a self report questionnaire on social support. It consists of different subscales 12. For this study, the subscale on perceived emotional support was used. This subscale contains five items, that can be scored on a four point Likert scale. Scores ranged from five to twenty, higher scores indicate a greater level of perceived emotional support 12. This questionnaire was completed at T0 only. Hospital Anxiety and Depression Scale (HADS) The HADS is a self reporting scale to indicate possible presence of anxiety and depression 13. This questionnaire was completed at T0, T1 and T2 and contains 14 items. Each item has four response options. The subscales for anxiety and depression consist of seven items each. Scores can range from zero to twenty-one for each subscale 13, 14. Higher scores indicate higher levels of anxiety or depression, respectively. Scores ≤7 are considered normal. Percentages of individuals with scores of ≥8 were calculated. Cronbachs т for the subscale scores ranged from 0.67-0.93 15. Self Efficacy Scale (SE24) The SE24 scale was focused on hearing impaired individuals in this study and was completed at T0, T1 and T2. This scale is used to evaluate the sense of control by means of five items. Four items can be scored on a five points Likert scale, one item can be scored on a four point Likert scale. Total scores range from five to twenty-four. A higher score reflects more sense of control. Crohnbach т ranges from 0.70-0.77 16. Impact of Event Scale (IES) The IES was completed at the T1 and T2. This scale evaluated the response on an event (getting acquainted with the genetic diagnosis). The scale consists of 15 items, which can be scored by a four point Likert scale. The 15 items can be divided into two subsets, avoidance and intrusion 17. Higher scores indicate more avoidance or intrusion due to the event. 164 | Chapter 4 Audiometry and genetic evaluation The data gathered with the questionnaires were complemented with audiometric results. Pure tone audiometry was performed according to current standards to determine hearing thresholds at frequencies of 0.25, 0.5, 1, 2, 4 and 8 kHz. The mean thresholds at 1, 2 and 4 kHz of the best hearing ear from the last available audiogram was noted as the pure tone average (PTA) for every individual. In all subjects Sanger sequencing of a specific deafness- associated gene was requested at T0. The selection of the gene to be tested was based on family history and audiometric phenotype. At T1 individuals were counseled about the positive (a mutation was identified) or negative (no mutation was identified) result of the genetic test. Statistic analyses Individuals were divided into two groups; a group with a positive genetic test result and a group with a negative test result. Demographic characteristics, including degree of hearing impairment and the results of the ICQ and ISR were compared between both groups by means of Chi square test for categorical variables and t-test for continuous variables. Continuous variables are presented by the median combined with the interquartile range (IQR). Linear mixed models for repeated measures were used for studying the differences between groups for each of the outcomes of the HADS subscales, SE24 and the IES subscales. The dependent variable was the genetic test result. The independent fixed variables were point of measurement (two levels: two and six weeks since baseline), group (Negative, Positive) and the baseline value. Individuals were treated as a random variable. Baseline-adjusted mean difference between groups at each measurement point with 95% confident intervals are presented. The statistical analysis was carried out by using IBM SPSS Statistics 20 (Chicago, IL) and SAS 9.0 for Windows (SAS Institute inc., Cary, NC).. Results Fifty-four individuals were eligible to be enrolled in this study. Four subjects did not complete the questionnaires at the first time point (T0). Two subjects were analyzed by means of next generation sequencing instead of single gene mutation analyses and were therefore excluded. Thus, 48 individuals were included in this study. In 11 of the 48 individuals (23%), a genetic cause of their hearing impairment was identified. Eight individuals were diagnosed with autosomal dominant inherited deafness (DFNA) based on mutations in COCH (DFNA9), two with DFNB8/10 (autosomal recessive inherited deafness (DFNB) based on mutations in TMPRSS3) and one with DFNA8/12 (TECTA). Psychosocial impact of a genetic diagnosis | 165 Table 1 Demographics and baseline characteristics. T0 T1 T2 Age Sex: - Female - Male Education: - ISCED 7 - ISCED 6 - ISCED 5 - ISCED 4 - ISCED 3 - ISCED 2 - ISCED 1 PTA (average 1, 2 and 4 kHz of the best hearing ear) Progression: - Yes - No Rehabilitation: - None - Hearing aid - Cochlear implant - Hearing aid and cochlear implant - SOLO History of psychiatric diseases: - Yes - No Children/ childrens wish: - Yes - No - Do not know Other hereditary diseases in the family - Yes - No - No answer Why do you pursue a genetic diagnosis? Treatment Prognosis Inheritance patterns ICQ (range 6-24) - Acceptation - Helplessness - Disease benefits ISR (range 5-20) Positive (n=11) Negative (n=37) 11/11 (100%) 8/11 (73%) 6 (55%) 54 (52-66) 37/37 (100%) 26 (70%) 20 (54%) 48 (36-58) 7/11 (64%) 4/11 (36%) 23/37 (62%) 14/37 (38%) 0/11 (0%) 0/11 (0%) 2/11 (18%) 2/11 (18%) 0/11 (0%) 6/11 (55%) 1/11 (9%) 73 (47-93) 9/37 (24%) 13 (35%) 0/37 (0%) 6 (16%) 3 (8%) 6 (16%) 0 (0%) 48 (32-71) 11/11 (100%) 0/11 (0%) 31/37 (84%) 6/37 (16%) 2/11 (18%) 7/11 (64%) 0/11 (0%) 2/11 (18%) 0/11 (0%) 14/37 (38%) 21/37 (57%) 1/37 (3%) 0/37 (0%) 1/37 (3%) 0/11 (0%) 11/11 (100%) 7/37 (19%) 30/37 (81%) 9/11 (82%) 1/11 (9%) 1/11 (9%) 28/37 (76%) 6/37 (16%) 3/37 (8%) 2/11 (18%) 8/11 (73%) 1/11 (9%) 9/37 (24%) 28/37 (76%) 0/37 (0%) 5/11 (45%) 6/11 (55%) 8/11 (73%) 21/37 (57%) 33/37 (89%) 26/37 (70%) 13 (7-14) 18 (13-21) 8 (7-11) 20 (19-20) 11 (8.5-14) 17 (12-18.5) 9 (7-12.5) 18 (14.5-20) ISCED (International Standard Classification of Education). Numbers between brackets are percentages or the median is presented with the upper and lower quartile between brackets. 166 | Chapter 4 Baseline characteristics All 48 subjects completed the questionnaires at T0. At T1, 34 individuals filled out the questionnaires. Eight of these received a positive result on genetic testing. Twenty-six individuals completed the questionnaires at T2, six of them had a positive genetic test result. As a consequence, 22 subjects were lost to follow up. Overall, the participants pursued a genetic diagnosis in order to be informed about treatment in 54% (26/48), about prognosis in 81% (39/48) and about inheritance patterns in 71% (34/48). Baseline characteristics were comparable for the positive and negative genetic test result group and are shown in Table 1. The only significant difference was observed for educational level. Individuals who received a negative result for genetic testing had a higher level of education than those with a positive result (p= 0.03). Illness cognition and social reliance Individuals of the positive and negative test result group were also comparable for each subscale of the ICQ. The mean score of all included subjects for helplessness was 11.2 and for disease benefits 9.7 (scoring range 6-24). This indicates that the hearing impaired individuals in this study experience helplessness toward their hearing impairment and do not experience disease benefits. The mean score for acceptance was 16.6 (scoring range 6-24). This indicates that the acceptance of hearing impairment is of an average level (figure 1A). Both groups also experienced similar levels of social support with a mean score for all subjects of 18.6. The scores B 25 25 20 20 15 15 Score Score A 10 5 5 0 0 – H s les elp 10 ss ne + – ce Ac + – e nc pta Di se b a se + – ts ef i en So c re ia l l ia + e nc Figure 1 Mean scores for the Illness Cognition Questionnaire (A) and the Inventory Social Reliance (B). The minus indicates the negative group, the plus indicates the positive group. The dashed lines indicate the possible scoring range. Psychosocial impact of a genetic diagnosis | 167 of the ISR range from 5-20, which indicates that the individuals in this study experience a great amount of perceived emotional support (figure 1B). Hospital Anxiety Depression Scale (HADS) The results of the HADS are shown for both the positive and negative test result group in table 2. Evaluation of the anxiety subscale shows some differences in both groups. The positive result group scores higher at T1, at T2 the scores have increased even more. Scores of the negative group at T2 have decreased and as a result the difference between both groups has increased at T2. This difference at T2 shows a trend towards significance (p=0.05; figure 2). Between the negative and positive result group no significant differences were found at both T1 and T2 for the depression subscale. In total, 14.6% of the cases scored 8 or higher on the anxiety subscale; 9.1% (1/11) of the positive group, 16.2% (6/37) of the negative group and 12.5% scored 8 or higher on the depression subscale (9.1% (1/11) of the positive group, 13.5 (5/37) of the negative group). When looked at the severely to profoundly hearing impaired individuals (PTA of ≥70 decibel hearing level) 13.3% (2/15) scored ≥ 8 on the anxiety subscale and 20% (3/15) scored ≥ 8 on the depression subscale. Self Efficacy 24 scale The Self Efficacy 24 Scale evaluates sense of control. No statistic significant difference was found between the scores for this sense of control of the positive and negative result group at T1 and T2. HADS-A 20 Negative Positive Score 15 10 5 0 T0 T1 T2 Figure 2 Anxiety level. Mean scores of the anxiety subscale of the HADS questionnaire at diagnosis and six weeks after in the positive and negative test result group. Dashed lines demonstrated the 95% CI’s. Dots and bars are means and standard deviation. 11 11 Positive 11 Positive 8 8 Positive 4.5 (0-20) 1 (0-28) 3.5 (0-10) 1.5 (0-29) 15.5 (9-20) 14.5 (8-21) 3 (0-7) 3.5 (0-11) 4,5 (1-12) 4 (0-14) Median 6 20 6 20 6 19 6 20 6 20 n T2 9 (0-24) 0 (0-33) 5.5 (0-22) 2 (0-27) 17.5 (13-21) 13 (3-20) 3 (1-10) 2.5 (0-9) 6 (1-8) 3.5 (0-11) Median -0.59 (-7.96-6.79) 3.82 (-4.13-11.76) 0.59 (-2.50-3.68) 0.61 (-1.20-2.43) -1.3 (3.36-0.76) Mean (95% CI) Difference at T1 between groups 0.87 0.33 0.70 0.49 0.20 p -1.90 (-9.58-5.77) -1.03 (-9.38-7.32) -1.88 (-5.13-1.38) -1.21 (-3.27-0.84) -2.3 (-4.64-0.05) Mean (95% CI) Difference at T2 between groups 0.61 0.80 0.24 0.23 0.05 p Number of participants in each group for each time point (T0, T1, T2). Median scores of each questionnaire of questionnaire subscale, with the lowest and highest score between brackets. Differences with the 95% confidence interval between brackets and p-values for the Hospital Anxiety and Depression Scale (HADS) subscales, Self Efficacy 24 (SE24) questionnaire and the Impact of Event Scale (IES) subscales between groups over time 26 Negative IES - Intrusion 26 8 26 8 26 8 26 Positive 17 (11-22) 15 (0-23) 4 (0-9) 3 (0-13) 3 (0-10) 4 (0-14) Negative IES – avoidance 37 Negative SE24 37 Negative HADS - depression 37 Positive n Median n Negative HADS - anxiety T1 T0 Table 2 Difference between groups over time. 168 | Chapter 4 Psychosocial impact of a genetic diagnosis | 169 Impact of Event Scale Overall, scores of IES were low. This means that either a positive or negative genetic test result does not lead to much avoidance or intrusion. The differences at T1 and T2 for both subscales were not statistically significant between the positive and negative result group. Discussion Currently, efficacy of genetic testing for hearing impairment is much higher than 10 years ago. Little is, however, known on the impact of such a diagnosis on psychological well being of hearing impaired individuals. The psychological impact of receiving a positive or negative genetic test result was evaluated in 48 individuals in this exploratory study. These individuals pursued a genetic diagnosis in order to be informed about treatment in 54%, about prognosis in 81% and about inheritance patterns in 71%. Receiving a positive result on genetic testing was not associated with an increase in psychological distress in the short term. Only a trend towards a significantly higher anxiety level in the positive genetic test result group was found. Comparison to other studies on the impact of a genetic diagnosis The study by Palmer et al. presented opposite findings compared to the current study. Patients who received a negative genetic test result had significantly higher anxiety levels after six months when compared to patients who received a positive test result (n= 183) 5. The effect demonstrated by Palmer et al. is also the opposite of other studies and remains present for a longer period of time 5, 7. A systematic review by Vansenne et al. demonstrated that a positive genetic test result might first lead to an increase in psychological distress levels that, however, vanishes over time 7. In the present study, we only evaluated short term effects of genetic testing. The result of the current study is similar to the outcome of the study by Reichelt et al. 18. This study investigated females who were presymptomatically tested on mutations in BRCA1, involved in increased susceptibility for ovarian and breast cancer. Receiving a positive genetic test result of a BRCA1 mutation was not associated with an increase in psychological distress. Reichelt et al. therefore stated that receiving a genetic diagnosis might be less distressing than getting the diagnosis of cancer itself. Considering the low scores on the impact of event scale in the current study, a similar consideration might be applicable to hereditary hearing impaired individuals. It could be hypothesized that receiving the diagnosis and dealing with sensorineural hearing impairment might increase levels of distress far more than receiving the genetic diagnosis that might explain the hearing impairment. 170 | Chapter 4 Attention to psychological distress In the current study, many individuals added personal information to the questionnaire, stating their appreciation for the attention to their psychological well being as a hearing impaired individual. Kvam et al. demonstrated that anxiety and depression occur significantly more in the hearing impaired population compared to the normal hearing population 19. Carlsson et al. demonstrated that 31.2% and 22.5% of the severely to profoundly hearing impaired patients score above the cutoff point of 8 on the HADS for anxiety and depression, respectively. The Swedish reference population scored in 12% and 9% of the cases above the cutoff point of 8 for anxiety and depression, respectively 20. In comparison, the present group of individuals has scores of ≥ 8 in 13.3% and 20% of the cases for anxiety and depression, respectively for the severe to profound hearing impaired individual. The percentage for depression is comparable to the one reported by Carlsson et al. This indicates that counselors on genetic testing for hereditary hearing impairment need to be aware of a possible increased level of psychological distress in individuals seeking a genetic diagnosis for their hearing impairment. In order to screen for psychological distress, a questionnaire focusing on these problems may be useful. As there were no significant differences in psychological distress between the positive and negative genetic test result group over time, this screening can take place at the first visit to the outpatient clinic. In 2013, Esplen et al. described the Genetic Psychological Risk Inventory (GPRI) that aims to identify subjects at risk for psychological distress after genetic testing for adult onset hereditary diseases 21 . This inventory is a useful screening tool to implement in the first visit. Dominant versus recessive inheritance patterns A possible explanation for the lack of difference in psychological impact between a positive and a negative diagnosis might be the large number of individuals with an autosomal dominantly inherited type of hearing impairment within this study. It can be assumed that patients with a dominantly inherited hearing impairment know a lot about the phenotype of their hearing impairment, based on the experiences of their relatives. They also might expect the genetic diagnosis more than subjects with a recessive type of inheritance. This study only included two subjects with autosomal recessive hearing impairment. Only one of them completed the questionnaires at T1 and T2. Therefore, the conclusion on the impact of a genetic diagnosis in DFNB patients remains more unclear. Study limitations and further recommendations One of the limitations of this study is the small number of subjects. This small number is due to the fact that this study was carried out at the time that testing a Psychosocial impact of a genetic diagnosis | 171 panel of hearing impairment genes by whole exome sequencing (WES) was being implemented in our clinic. The implementation of targeted exome sequencing as a routine diagnostics in our out-patient clinic has completely changed our protocol and single gene analysis is currently only performed in selected cases with a very directive phenotype such as is seen in DFNA9 and GJB2 is tested due to the large prevalence of DFNB1. In all other cases, the deafness gene panel test by WES is the preferred first choice of diagnostics. Therefore, the number of individuals who were suitable for single gene genetic testing decreased. The gene panel test entails other factors that might influence the psychological status of a hearing impaired individual, such as finding a mutation in a gene associated with syndromic hearing impairment. This study needs to be repeated for individuals tested by whole exome sequencing in order to state something on the psychological distress in individuals who undergo whole exome sequencing to find the cause of their hearing impairment. When this study is repeated it should be considered to include hearing impaired subjects who do not request genetic testing in order to exclude a possible selection bias. In this study only subjects eligible for genetic testing were included which could lead to a selection bias. In addition to the small number of individuals that could be included, quite a number of individuals were lost to follow up. A possible explanation could be the small window of time in which individuals were allowed to complete the questionnaires of T1. This study only focused on non-syndromic hearing impairment. In several syndromic types of hearing impairment, the additional symptoms occur at a later age 22. The knowledge that these additional symptoms can eventually occur might cause more psychological distress. Therefore, the results of this present study might not be applicable to individuals who are genetically tested for syndromic hearing impairment. Conclusion This pilot study evaluated the psychological impact of a genetic diagnosis of hereditary hearing impairment in 48 individuals. There was no difference in psychological impact between a positive or negative genetic test result and the test results did not significantly influence psychological well being of the participants in a positive or negative way. This study also demonstrated that increased levels of depression can be observed in severe to profound hearing impaired individuals that visit the out-patient clinic for a genetic diagnosis. 172 | Chapter 4 Further research is needed to evaluate the psychological impact of a genetic diagnosis of hereditary hearing impairment and of the impact of hearing impairment itself. Testing of a panel with known deafness- associated genes has led to an increased percentage of positive genetic diagnosis in our clinic. The present study, therefore needs to be repeated in individuals who are tested by the hereditary hearing impairment gene panel test. Psychosocial impact of a genetic diagnosis | 173 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Morton CC, Nance WE. Newborn hearing screening--a silent revolution. The New England journal of medicine. 2006;354(20):2151-64. Van Camp G, Smith R. Hereditary Hearing Loss Homepage. 2014 [4-12-2014]. 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Mental health in deaf adults: symptoms of anxiety and depression among hearing and deaf individuals. Journal of deaf studies and deaf education. 2007;12(1):1-7. Carlsson PI, Hjaldahl J, Magnuson A, et al. Severe to profound hearing impairment: quality of life, psychosocial consequences and audiological rehabilitation. Disability and rehabilitation. 2014:1-8. Esplen MJ, Cappelli M, Wong J, et al. Development and validation of a brief screening instrument for psychosocial risk associated with genetic testing: a pan-Canadian cohort study. BMJ open. 2013;3(3). Kochhar A, Hildebrand MS, Smith RJ. Clinical aspects of hereditary hearing loss. Genetics in medicine: official journal of the American College of Medical Genetics. 2007;9(7):393-408. 5 Discussion and Conclusion Discussion and conclusion | 177 The aim of this thesis was to describe new genotype-phenotype correlations for autosomal recessively inherited hearing impairment in order to improve counseling and to gain more knowledge on the effect of different (mutated) genes on cochlear performance. Clinical characteristics of known genotype-phenotype correlations were first summarized in a general review on autosomal recessively inherited hearing impairment in chapter 1.2. The first described genotype-phenotype correlation in this thesis are those of DFNB18B and DFNB84B. In chapter 2.1, phenotypes of patients with mutations in OTOG and OTOGL were compared. Previous studies concluded that OTOG and OTOGL are comparable in terms of expression and that otogelin and otogelin-like have structural similarities. The present study demonstrated that phenotypes resulting from mutations in these two genes are also similar. The second genotype-phenotype correlation described in chapter 2.2, focused on mutations in USH1G. Mutations in USH1G were prior to this study only linked to syndromic hearing impairment, i.e. Usher syndrome type 1, or atypical Usher syndrome. The patients in this study, however, presented with moderate hearing loss, and in addition, normal vestibular and ophthalmologic function was found. Therefore, this is the first description of nonsyndromic hearing impairment based on mutations in USH1G. In chapter 2.3, the third genotype-phenotype correlation was the description of a phenotype resulting from recessive mutations in a novel deafness gene: CLIC5. Besides hearing impairment and vestibular dysfunction, also early indications of a possible mild renal dysfunction were found. In the third chapter of this thesis, the genotype-phenotype correlation of DFNB1, the most common autosomal recessively inherited type of hearing loss, was expanded by further examining temporal bone imaging and assessing vestibular function. It was concluded that DFNB1 might not be associated with specific temporal bone anomalies or with vestibular dysfunction. In chapter 4, we focused on the psychological impact of receiving genetic results concerning hearing impairment. From this study it was concluded that receiving a genetic diagnosis on hearing impairment does not affect psychological distress levels. Therefore, genetic evaluation of hearing impairment can be offered to each hearing impaired patient. Physicians in general should also be conscious of the enhanced susceptibility to psychological stress in severe to profound hearing impaired patients. The results of these studies contribute to counseling of patients with hereditary hearing impairment and their families. These studies are also essential for future studies on therapy for hereditary hearing impairment, because they provide insight in the natural progression of the disease. 178 | Chapter 5 From animal models to clinical consequences Mouse models When a novel human deafness gene is identified, knowledge on the function of the encoded protein in the cochlea is often obtained from animal models. Different animal models are used to study hereditary hearing impairment e.g.: mice, guinea pigs and zebrafish. Most of the knowledge on hereditary hearing impairment is, however, based on studies in mouse models. A candidate human deafness gene can be studied in animal models by performing gene targeted mutagenesis to create a knock-out mouse model. Not only the expression of the mutated gene can be studied in vivo in an animal model, but also the functional consequences can be studied for example by testing auditory brainstem responses 1. Since otogelin was found to be part of the tectorial membrane in mice 2, we hypothesized a conductive cochlear hearing loss in patients with hearing loss resulting from mutations in OTOG. However, psychophysical testing did not confirm this hypothesis (chapter 2.1). On the other hand, Clic5 knockout mice displayed, besides hearing loss, also dysfunction of various other organs. Therefore, the DFNB102 patients were screened for other, subclinical organ dysfunctions and, the first signs of nephropathy were discovered. Without the clues from the mouse model, this would have remained unnoticed until the first symptoms would appear. Now these patients can be followed up and early interventions are possible when necessary (chapter 2.3). In chapter 3, the results of vestibular testing in DFNB1 patients are correlated to histological observations in connexin 26 (GJB2) and connexin 30 (GJB6) knock-out mice. From connexin 30 knock-out mice it can possibly be concluded that insufficient numbers of gap junctions may lead to saccular hair cell death 3. The human sacculus can be evaluated in vivo by means of measuring cervical vestibular evoked myogenic potentials (cVEMP). These cVEMPs could not be evaluated due to the retrospective character of the study in chapter 3. However, a recent study demonstrated signs of saccular dysfunction in DFNB1 patients 4. In conclusion, it can be stated that mouse models are very valuable to study the function of proteins in the cochlea and other organs, when these proteins are encoded by newly identified human deafness- associated genes. The translation to humans, however, should be made very carefully and supported by clinical audiometric findings. Phenotype evaluation and number of affected patients When a phenotype description is based on a large group of patients, it is considered to be more precise than when it is substantiated by a small number of cases. This makes research on autosomal recessively inherited hearing impairment in the Discussion and conclusion | 179 Western world with small families and low numbers of affected individuals difficult. In order to make phenotype descriptions of autosomal recessive hearing loss more accurate and to uncover possible variability, larger numbers of patients are needed. Variability of phenotypic effect of mutations The phenotypic effect of different mutations in a gene can be highly variable. This is, for example, clearly demonstrated in DFNB1. Snoeckx et al. performed a multicenter study on DFNB1 patients and showed that phenotypes of biallelic truncating mutations are more severe than those of biallelic non-truncating mutations 5. Studies describing novel deafness- associated genes often include different families with different mutations in the same deafness gene. The numbers of patients with autosomal recessive hearing loss based on a specific gene are usually small. Therefore, it is difficult to establish robust genotype-phenotype correlations. The variation in phenotypes caused by mutations in the same gene is demonstrated in chapter 2.2. In this chapter, the examined family with causative mutations in USH1G was compared to a family studied earlier by Weil et al. 6. Both families, with similar genotypes, present with completely different phenotypes: syndromic as well as nonsyndromic. Modifier genes Differences in phenotypes can even exist between patients with the same genotype. Weegerink et al. described, for example, that the age of onset within DFNB8/10 families could differ by more than ten years 7. This heterogeneity can possibly be explained by effects induced by, amongst others, modifier genes. When it comes to phenotypic expression of a mutation, the modifier gene can enhance the mutant phenotype, or suppress it. Modifier genes may therefore lead to more severe or milder hearing impairment, respectively 8. Research into modifier genes and associated mutations will provide more insight in the biological pathways influenced by the primary mutated gene in the inner ear. In order to identify these modifier genes, large numbers of patients with comparable genotypes need to be studied. When there is a large variability in phenotype, the whole exome sequencing (WES) data of the worst and best hearing individuals can be compared. Since WES is emerging as a diagnostic tool, it will facilitate identification of modifier genes. The first dominant modifier gene is located at DFNM1. In a Pakistani family with eight hearing impaired family members affected by DFNB26, seven non-affected family members appeared to display the same haplotype as the affected subjects. During linkage analysis the DFNM1 locus was defined 9. However, the identity of the DFNM1 modifier is still elusive. Autosomal recessively inherited modifier genes have not been identified thus far. 180 | Chapter 5 Environmental factors Hearing impairment can also be influenced by environmental factors. The most important environmental factor in hearing is noise exposure. Daily exposure to 85 dB SPL (sound pressure level) or more may lead to noise induced hearing impairment 10 . Genetic factors can predispose to hearing impairment caused by noise exposure. This is demonstrated in several mouse models 11. Mutations in TRPV4 may enhance susceptibility to noise induced hearing impairment . This might explain the large variability in hearing thresholds within a family known with hearing impairment due to mutations in TRPV4 12. Exposure to different types of chemicals may also lead to hearing impairment. No consensus has been reached yet on the influence of tobacco and alcohol on hearing. Another familiar environmental factor is ototoxic medication; loop diuretics, chemotherapy and aminoglycoside antibiotics are known to cause hearing impairment 11. The latter is also associated with genetic predisposition. Some mitochondrial mutations are, for example, known to enhance the susceptibility to hearing impairment caused by the use of aminoglycoside antibiotics. Mutations in MT-RNR1 are known to cause this predisposition 13. Therefore, testing for known mutations associated with susceptibility to aminoglycosides should be performed. Clinicians should be aware of this susceptibility before starting treatment with aminoglycoside antibiotics. For example when the mother of the patient is hearing impaired after antibiotic use, since mitochondrial mutations inherit maternally. Improving diagnostics in hereditary hearing impairment Genetics Due to the heterogeneous nature of hereditary hearing loss, it is difficult to select the right candidate gene for diagnostics based on clinical features. The introduction of WES has changed this process since all exons are screened for mutations and candidate gene selection is no longer necessary. Phenotype driven testing is in the Netherlands often replaced by analyzing only known deafness- associated genes from the WES data (targeted exome sequencing). When the mutated gene cannot be found by this analysis, targeted exome sequencing can be followed by opening the exome. Up to 60% of cases of hereditary hearing impairment are currently solved by WES 14. It can, therefore, be expected that phenotype driven genetic testing will become less valuable than previously and will only be applied to clearly distinguishable phenotypes, such as amongst others DFNA9 (COCH). Due to its high prevalence, DFNB1 (GJB2/ GJB6) should always be tested in non syndromic recessive hearing impairment or isolated cases 15. Discussion and conclusion | 181 Novel exome and whole genome sequencing technologies are still evolving, the techniques become faster and cheaper. One of these new techniques is, for example, nanopore sequencing. With this method, individual DNA strands pass through tiny protein pores. While these strands pass, the changes in electrical current are measured 16, 17. Up to 512 DNA molecules can be read simultaneously. This is a single molecule method that does not require fluorescent labeling. This will improve the speed of sequencing and will reduce costs 18. These new techniques produce data faster and in larger quantities than ever before. These new techniques also have their disadvantages. Since the amount of data keeps increasing, data storage needs to improve in order to handle the large amounts of sensitive data. This type of data is interesting for different people and therefore should be carefully managed in order to prevent genetic discrimination 19. Data interpretation is another issue. Variants of unknown significance and secondary findings should be handled with care. In addition, the patient and its counselor need to be aware of what the patients wants and must know 20-22. For example, in case of incidental findings on predisposing mutations associated with hereditary breast cancer or spinal muscular atrophy. Imaging in hereditary hearing impairment When the genetic defect has been determined, the next step is to evaluate whether there is an effect on the inner ear structures. The resolution of magnetic resonance imaging and computed tomography scans is improving, but up until now it is still not possible to reveal pathogenic pathways in the cochlea on a micro scale. Currently, the only way to visualize the microanatomy of the cochlea is by histopathology. Optical coherence tomography is an imaging technique that allows evaluation of the microanatomy of the cochlea in vivo. In ophthalmology this technique is already extensively used and depicts, amongst others, structural and functional characteristics of the complete eyeball 23 (chapter 2.2). Therefore, this is also an interesting research topic in the inner ear 24, 25. Optical coherence tomography can determine structures with a resolution between 2 and 20 +m. The principle of this technique is analogous to the principle of ultrasound. Ultrasound is, however, based on reflection of acoustic stimuli from biological tissue layers, while optical coherence tomography is based on the reflection of light. A disadvantage thus far is that direct sight on the cochlea was necessary in mouse models to retrieve good images of the microstructures 26. Imaging of the cochlea on a micro scale in vivo would reveal insight into the effect of genetic defects on structural dysfunction. For example, in mice defective for otogelin, detachment of the otoconial membrane in the utricle and saccule is seen. Since DFNB18B (OTOG) patients demonstrated vestibular hyporeflexia it can be expected that the otoconial membrane in humans is also defective. 182 | Chapter 5 With in vivo imaging of the cochlea this hypothesis could be directly confirmed or rejected. Improving audiometric evaluation Patients not only want to be informed on the cause of their hearing loss, but usually also prefer to know how their hearing will function in the future. For this purpose, expanding knowledge on psychophysical results is necessary. In Nijmegen, several families with hereditary hearing impairment have been evaluated by means of the Nijmegen psychophysical test battery. Unfortunately, no indisputable conclusion could be drawn from the studies on sensory types of hearing impairment since the numbers of tested patients were small and the results demonstrated variability between patients from the same families 27-31. On the other hand, the Nijmegen psychophysical test battery did successfully indicate the cochlear conductive type of hearing impairment in DFNA8/12 (TECTA) and DFNA13 (COL11A2) patients 32, 33. The test battery proposed by the HEARCOM project could be a solution for the small number of patients. One of the goals of the HEARCOM project was to create an auditory profile for each patient. In order to do so, a test battery was composed which could be used in several European countries. This test battery contains the Acalos test to determine loudness perception and the combined F and T-test for frequency resolution and temporal acuity. To evaluate speech reception the SRT in quiet, stationary and fluctuating noise and the MATRIX-OLSA test are used 34. When the same tests are used internationally, larger numbers of patients can be evaluated and compared. Hopefully, these kind of studies lead to more knowledge on the auditory phenotype. Ultimate goal: normal hearing Normal hearing is the ultimate wish of every patient with hereditary hearing loss. This wish can currently not be fulfilled and therefore the main focus lies on rehabilitation by (implantable) hearing devices. Another treatment option that is explored is based on preservation and restoration of dysfunctional parts of the cochlea. Rehabilitation Hearing aids The first choice of rehabilitation in patients with a mild to moderate sensorineural hearing loss is a hearing aid. Remarkably, research on hearing aids in hereditary hearing-impaired patients is rather scarce. Research on outcomes of different hearing aid settings in different types of hearing impairment can enhance fitting Discussion and conclusion | 183 of hearing aids in hereditary hearing impaired patients. For example, patients with a cochlear conductive hearing impairment can be predicted to benefit more from a linear setting of their hearing aids than patients with a sensory type of hearing impairment. A cochlear conductive hearing loss, such as DFNA13 (COL11A2) and DFNA8/12 (TECTA), is associated with a shifted curve during loudness scaling 32, 33. Patients with sensorineural hearing loss present with a steeper than normal curve, which is distinctive for recruitment. To overcome recruitment, hearing aids are set with a compressed setting 35. Since DFNA13 (COL11A2) and DFNA8/12 (TECTA) patients present with sensorineural hearing loss during standard audiometric evaluation, their hearing aids are set to a compressed setting. While patients without recruitment (and a (cochlear) conductive hearing loss) might benefit more from a linear setting. More research is therefore needed on the translation of cochlear dysfunction and audiometric profiles into hearing aid adjustments. Cochlear implantation A number of correlation studies on performance after cochlear implantation in patients with hereditary hearing impairment have been published so far. For example, Vermeire et al. evaluated speech perception after cochlear implantation in DFNA9 patients. When compared to other patients with adult onset sensorineural hearing loss, DFNA9 patients performed equally or even better after cochlear implantation. Since DFNA9 patients demonstrate severe auditory neuronal damage, these results were not expected 36. Performance after cochlear implantation is influenced by multiple factors. The most important factors are age at implantation, inner ear malformations and duration of hearing impairment before implantation 37-39. Therefore, drawing conclusions on the effect of the type of hereditary hearing impairment on postoperative performance is rather difficult. This is particularly difficult because the numbers of patients in these studies are still relatively low. In the mean time, cochlear implantation keeps improving considering every aspect. One of these aspects is electro-acoustic stimulation (EAS). EAS combines acoustic and electric stimulation in the same ear and yields better performance than electric stimulation only. EAS also improves results of speech perception, hearing in noise and music appreciation 40. In order to preserve residual hearing after cochlear implantation different aspects of the procedure have been studied. Different characteristics of hearing impairment influence preservation of residual hearing after implantation. In congenital hearing loss, more residual hearing is retained after implantation than in acquired or idiopathic hearing loss. Progressive hearing impairment is less likely to retain residual hearing after implantation 41. Therefore, accurate genotype-phenotype correlations are important in counseling of cochlear implant candidates on the expectations of a cochlear implant (CI). 184 | Chapter 5 A shallow insertion depth would reduce trauma to the cochlea, and therefore give better results concerning residual hearing 40. In progressive types of hearing impairment this should be a point of interest since the possibility of acoustic stimulation will diminish over time. In such cases, a deeper insertion depth, with a longer, atraumatic electrode should be considered. In this respect, accurate genotype-phenotype correlations are also important. Initially, EAS seems a good option in DFNB8/10 patients, since hearing thresholds of the lower frequencies remain well at first. However, at a later stage hearing loss progresses7 and complete electric stimulation is necessary. Insertion of the electrode can provoke an inflammatory response, which can further damage the cochlea. Administration of topical or systemic steroids perioperatively, reduces the inflammatory intracochlear response and leads to better preservation of residual hearing 41, 42. Besides preservation of residual hearing, improving the coating of the electrode is also an interesting field of research, since it can be used as a drug delivery system. Maintenance or stimulating of the outgrowth of spiral ganglion cell neurites by neurotrophic factors is of special interest 43, 44. This could also be interesting in hereditary hearing impairment types that involve the spiral ganglion, such as DFNB8/10 45. Increasing knowledge on proteins encoded by deafness- associated genes and their function, or dysfunction in hearing impaired individuals, might provide insights in new factors to investigate for their positive effect when applied via the coating of electrodes. Intracochlear therapy Hearing with hearing aids is not a perfect solution. Restoration of normal hearing without having to use a hearing aid is the ultimate goal. The first steps are being made towards this prospect. Different therapies are currently explored like gene therapy, stem cell therapy and regeneration of cochlear structures. Gene therapy With genetic therapy a disease is treated by genetic molecules. Specific DNA or RNA is administered by a vector into the target cells in order to compensate for loss-of-function mutations, interfere with gain-of-function mutations or suppress a dominant negative mutation. Gene therapy has already been performed in mice with hereditary hearing impairment. Mice that do not express vesicular glutamate transporter 3 (Vglut3) are congenitally deaf. Normal synaptic transmission cannot be carried out without a glutamate transporter. Vglut3 was introduced in the inner hair cells of Vglut3 knockout mice by means of an adeno-associated viral vector encoding the wild type Vglut3. As a result, auditory brain stem responses Discussion and conclusion | 185 normalized and this effect remained for at least some weeks. Another interesting result is that the delivery technique also influenced the results since a cochleostomy was found to be more traumatic than delivery through the round window membrane 46. Splice site mutations can also be corrected by means of genetic therapy. In mice with splice site mutations in Ush1c hearing impairment and vestibular dysfunction occur. Antisense oligonucleotides can be used to correct defective splicing. In Ush1c.216A knock–in mice, antisense oligonucleotides were administered intraperitoneally. When this administration took place early in the development of the inner ear, hearing and vestibular function could be retained 47. In humans, SLC17A8 codes for VGLUT3 48 and mutations in USH1C can cause Usher syndrome and nonsyndromic hearing loss 49, 50. However, before these therapies are applicable in humans, some difficulties have to be overcome. For the strategies mentioned above the different routes of delivery are outlined in table 1. Many factors such as safety, immunogenicity, expression regulation and long term efficacy need to be addressed before genetic therapies can be broadly applied in humans 51. Autosomal recessively inherited hearing impairment often has a congenital onset. This congenital onset occurs due to partial failure of development of the cochlea. Development of the human ear is completed in utero and this means that genetic therapy should also be applicable in a later stage, since treatment in utero is fairly impractical 54. Other organs, such as the eye, are more suited for gene therapy research. In ophthalmology, gene augmentation therapy has been studied extensively in retinal degeneration disorders and positive results in humans have been published already 55-57. A phase 3 clinical trial is currently initiated 58. This expertise on genetic therapies in retinal degeneration disorders may well provide new insights applicable to disorders of the cochlea. Stem cell therapy Another future treatment option could be stem cell therapy. Stem cells are introduced into the cochlea with the aim that these cells differentiate into cells specific to the inner ear (e.g. hair cells, auditory neurons) once they are in the right environment 59. Differentiation into hair cells encounters still some difficulties. A large amount of the stem cells transplanted into the cochlea do not integrate into the organ of Corti and therefore, do not develop into hair cells 60. However, some promising results have been achieved with human fetal auditory stem cells (hFASCs). These specific stem cells could differentiate into auditory neurons in vitro. Eventually, these stem cells were transplanted into the cochlea of gerbils with an auditory neuropathy. This improved the auditory function substantially 61. Which cells are infected? Mostly vestibular cells Inner and outer hair cells Supporting cells Spiral ganglion cells Cells in the vestibule and scala vestibuli All cochlear cells Cells in the: endolymphatic sac and duct; Vestibular end organs Stria vascularis; Reisners membrane; Hensen cells Route of delivery Intratympanic injection Round window sponge Injection through a canalostomy (posterior semicircular canal) Round window injection Oval window injection Injection through a cochleostomy Endolymphatic sac injection Hearing outcome was not reported. Higher incidence of sensorineural hearing loss - - Transduction is variable Low uptake in inner ear due to diffusion through the round window Low uptake in inner ear due to diffusion through the round window Disadvantage of the technique Table 1 Different routes of delivering therapeutic molecules into the cochlea 52, 53. 186 | Chapter 5 Discussion and conclusion | 187 Regeneration therapy Humans do not have the capacity to regenerate hair cells, however, regeneration of hair cells is seen in, amongst others, avian and zebrafish’ inner ear. Atoh1 has been demonstrated to be regulating the behavior of progenitor cells 62. In human, ATOH1 is a gene of great interest for regeneration therapy since it codes for a transcription factor necessary for hair cell development. Different studies have already demonstrated that delivery of Atoh1 in mice by an adeno-associated virus can restore auditory and vestibular hair cells and therefore, can restore loss of function 54. Some remaining cells need, however, to be present in order to restore hair cells. This is not possible from undifferentiated epithelium 63. The difficulties of genetic therapy mentioned above also apply to this type of regeneration therapy. Future directions In different paragraphs of this discussion it is indicated that larger numbers of patients are needed to determine genotype-phenotype correlations more accurately and to evaluate and identify possible influences of environmental factors and modifiers genes. In addition, larger numbers of patients with hereditary hearing impairment need to be evaluated by psychophysical testing in order to be able to draw conclusions on the type of hearing loss. Also, more patients are to be evaluated to establish a correlation between performance with a CI or hearing aids and genetic types. Since the numbers of patients with defects in a specific gene for autosomal recessive nonsyndromic hearing impairment are small for most of the deafnessassociated genes, international collaborative studies with standardized evaluation protocols are necessary to establish correlations between genetic defects, the deafness phenotypes and efficacy of rehabilitations methods. Only then, counseling on hereditary hearing impairment can be improved and therapeutic strategies evaluated to reach optimal results for individuals with hereditary hearing impairment in the future. 188 | Chapter 5 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Friedman LM, Dror AA, Avraham KB. Mouse models to study inner ear development and hereditary hearing loss. The International journal of developmental biology. 2007;51(6-7):609-31. Simmler MC, Cohen-Salmon M, El-Amraoui A, et al. Targeted disruption of otog results in deafness and severe imbalance. 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Chen W, Jongkamonwiwat N, Abbas L, et al. Restoration of auditory evoked responses by human EScell-derived otic progenitors. Nature. 2012;490(7419):278-82. Cafaro J, Lee GS, Stone JS. Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration. Developmental dynamics : an official publication of the American Association of Anatomists. 2007;236(1):156-70. Izumikawa M, Batts SA, Miyazawa T, et al. Response of the flat cochlear epithelium to forced expression of Atoh1. Hearing research. 2008;240(1-2):52-6. Discussion and conclusion | 191 6 Summary / Samenvatting 6.1 English summary Summary / Samenvatting | 197 Summary This thesis starts with a general introduction on hereditary hearing impairment. Different topics are discussed, from the basics of sound and anatomy and physiology of the human ear to characteristics of hearing impairment and genetic testing. A review that summarizes all currently known genes involved in autosomal recessively inherited types of hearing impairment is found in chapter 1.2. The review concludes that genotype-phenotype descriptions in deafness gene identification studies often lack precise information on audiovestibular characteristics. Chapter 2 describes several novel genotype-phenotype correlations. In chapter 2.1 the phenotypes of two novel human deafness- associated genes are described, OTOG and OTOGL. Seven hearing impaired patients of two families demonstrated mutations in OTOGL. In two other families six hearing impaired patients showed mutations in OTOG. A similar type of hearing impairment is found in patients with mutations in either one of the genes. In general, patients present with a mild to moderate hearing impairment and with a flat to downsloping audiogram configuration. Speech recognition scores remain fairly well (>90%). In one of the four families progression was seen after the second decade. In order to further characterize the hearing impairment, extensive audiometric tests were performed. The curves of loudness scaling at two kHz run steeper than normal hearing individuals and speech recognition scores in noise are worse than normal hearing individuals. These results indicate a sensorineural type of hearing impairment, instead of a intracochlear conductive hearing impairment. The latter could be expected, based on the involvement of otogelin and otogelin-like in the tectorial membrane. Previous studies on TECTA and COL11A2, which are expressed in the tectorial membrane, displayed an intracochlear conductive type of hearing impairment. In two families the vestibular function was assessed which demonstrated a hyporeflexia. Therefore, it is concluded from this chapter that both novel deafness genes give rise to a similar phenotype when mutated. In chapter 2.2, a novel non-syndromic hearing impairment is linked to mutations (c.310A>G, p.(Met104Val) and c.780insGCAC, p.(Tyr261Alafs*96)) in USH1G. Thus far, only Usher syndrome type I and atypical Usher syndrome were linked to mutations in USH1G. Usher syndrome type I is characterized by congenital, stable, severe to profound hearing loss, vestibular areflexia, and retinitis pigmentosa that has an onset before puberty. The phenotype described in this chapter displays an early onset hearing impairment with a downsloping audiogram configuration. The hearing impairment only progresses slightly. Extensive examination of the eyes and vestibulum did not display any abnormalities. 198 | Chapter 6 This non-syndromic character of this hearing impairment is assigned to the non-truncating mutation (c.310G>A). Modeling of this mutation predicted only a surface change and not a structural change of the SANS protein. In chapter 2.3, the genotype-phenotype correlation of two siblings from a consanguineous Turkish family is described. Both patients presented with early childhood onset of hearing impairment. Hearing impairment progressed quickly from mild to severe to profound before the second decade. Initially, motor milestones were normal, however, also vestibular function deteriorated to vestibular areflexia over time. Additionally, in one of the patients mild renal dysfunction is noted. This phenotype is linked to a homozygous nonsense mutation c.96T>A (p.(Cys32X)) in CLIC5. In this chapter it is demonstrated that CLIC5 is expressed in many human tissues. However, besides the mild renal dysfunction no additional symptoms have been found. Mutation analysis for CLIC5 was applied to 213 patients with autosomal recessive non-syndromic hearing impairment from mostly Dutch and Spanish origin. No additional pathogenic variants were revealed. CLIC5 is therefore a novel deafnessassociated gene, however, mutations in this gene are not a common cause of autosomal recessive hearing loss in the Dutch and Spanish populations. Chapter 3 is aimed to expand the phenotype description of the most prevalent type of hereditary hearing impairment, DFNB1. The audiometric phenotype is very heterogeneous and extensively described in literature. Less is known on temporal bone anomalies and vestibular function and in literature there is no consensus on these topics. In this chapter, 44 DFNB1 patients were evaluated for their vestibular function and/ or temporal bone imaging results. Five patients complained of balance problems or displayed delayed motor milestones. However, all patients demonstrated a response during velocity step testing. The calorisation test results were within normal range bilaterally in 65% of cases and the video head impulse test was normal in all tested patients. Therefore, vestibular dysfunction is not correlated to DFNB1. In this retrospective study, saccular function was not evaluated by cVEMPs and it is therefore recommended to evaluate this in future studies. In 34.4% of the computed tomography scans one or more temporal bone anomalies were identified. A variety of anomalies was found and all presented in small numbers, therefore, none seemed convincingly connected to a specific DFNB1 genotype. Summary / Samenvatting | 199 In chapter 4 the focus is not on hearing impairment itself, but on the impact of a genetic diagnosis of hereditary hearing impairment. Forty-eight patients were asked to fill out the Hospital Anxiety Depression Scale, Impact of Event Scale, Self Efficacy 24, Illness Cognition Questionnaire and the Inventory for Social Reliance. General characteristics were collected by means of a self constructed questionnaire and reviewing patient files. The questionnaires were completed at three time points: before genetic testing, directly after counseling on the results and six weeks thereafter. There were no differences between patients who received a genetic explanation for their hearing impairment (positive result) and the patients who did not (negative result). This study also confirmed that hearing impaired individuals may present with an increased level of psychological distress. It is, therefore, recommended to pay special attention to the psychological well being of hearing impaired individuals, who present at an outpatient clinic to have the cause of their hearing impairment identified. A positive genetic test result, however, does not affect their psychological well being. No reasons in favor or against genetic testing on hereditary hearing impairment were found in this chapter. By means of this thesis a contribution is made to the knowledge of autosomal recessive hearing impairment. This is, however, only a small part of the research on hereditary hearing impairment. Expansion of the possibilities to find novel genes or mutations, to examine the cochlea or to treat hereditary diseases will eventually lead to the ultimate goal: normal hearing. 6.2 Nederlandse samenvatting Summary / Samenvatting | 203 Samenvatting Dit proefschrift begint met een algemene introductie over erfelijke slechthorendheid. Deze introductie beschrijft onder andere de basiskarakteristieken van geluid, de anatomie en fysiologie van het menselijk oor, de kenmerken van gehoorverlies en tot slot genetische diagnostiek. Hoofdstuk 1.2 bevat een overzicht van alle genen die op dit moment geïdentificeerd zijn voor autosomaal recessief gehoorverlies. Een van de conclusies van dit overzicht is dat artikelen die een nieuw doofheidsgen beschrijven vaak een grondige genotypefenotype beschrijving missen over de audiovestibulaire karakteristieken. Dit belemmert adequate counseling van patiënten met een zelfde vorm van erfelijk gehoorverlies. Hoofdstuk 2 bevat enkele genotype-fenotype correlaties van nieuwe doofheidsgenen. Hoofdstuk 2.1 beschrijft de fenotypes van twee nieuwe doofheidsgenen, OTOG en OTOGL. Zeven slechthorende patiënten uit twee families dragen mutaties in OTOGL. In twee andere families zijn er zes patiënten met gehoorverlies en mutaties in OTOG geïdentificeerd. Gehoorverlies met dezelfde karakteristieken werd in alle vier de families gevonden. Mild tot matig van ernst en met een vlak tot aflopende audiogramconfiguratie. Het spraakverstaan bleef redelijk goed (>90%). In een van de vier families werd progressie gezien na het twintigste levensjaar. Om het gehoorverlies nog verder te karakteriseren zijn psychofysica-testen uitgevoerd. De luidheidsschaling bij 2 kHz resulteerde in curven die steiler verlopen dan bij normaal horenden en daarnaast werden er ook slechtere spraakverstaan in ruis scores gevonden in vergelijking met normaal horende individuen. Deze resultaten wijzen op een sensorineuraal gehoorverlies, in plaats van een intracochleair conductief verlies. Dit laatste werd verwacht gezien de lokalisatie van otogelin en otogelin-like in de tectoriaal membraan. Eerdere studies hebben laten zien dat genen die tot uiting komen in de tectoriaal membraan juist een intracochleair conductief verlies veroorzaken. De vestibulaire functie werd in twee families onderzocht en dit liet een hyporeflexie van het vestibulaire orgaan zien. De conclusie van het onderzoek uit dit hoofdstuk is dat mutaties in beide nieuwe doofheidsgenen eenzelfde fenotype veroorzaken. Hoofdstuk 2.2 beschrijft een niet syndromaal gehoorverlies dat veroorzaakt wordt door mutaties (c.310A>G, p.(Met104Val) en c.780insGCAC, p.(Tyr261Alafs*96)) in USH1G. Tot dusver zijn alleen Usher syndroom type I en het atypische Usher syndroom geassocieerd met mutaties in USH1G. Usher syndroom type I wordt gekenmerkt door een congenitaal, stabiel, ernstig tot zeer ernstig gehoorverlies, vestibulaire areflexie en retinitis pigmentosa die begint voor de puberteit. Het in 204 | Chapter 6 dit hoofdstuk beschreven fenotype laat een gehoorverlies zien dat vroeg in het leven tot uiting komt en een aflopende audiogramconfiguratie heeft. Het gehoorverlies is maar matig progressief. Uitgebreid onderzoek van de oculaire en vestibulaire functie lieten geen afwijkingen zien. Het niet-syndromale karakter van dit gehoorverlies wordt toegeschreven aan een niet-truncerende aminozuursubstitutie (c.310G>A). Een bio-informatisch model van deze mutatie voorspelde namelijk een oppervlakte verandering en geen structurele verandering van het SANS eiwit waarvoor USH1G codeert. Hoofdstuk 2.3 beschrijft een genotype-fenotype correlatie van een broer en zus uit een consanguine Turkse familie. Beide patiënten lieten een gehoorverlies zien dat vroeg in de kindertijd begint. Het gehoorverlies verslechterde van mild naar ernstig tot zeer ernstig voor het tiende levensjaar. De initiële motorische ontwikkeling was normaal, maar de vestibulaire functie verslechterde in de loop der tijd tot vestibulaire areflexie. Aanvullend werd er in één van de patiënten een milde nierfunctiestoornis gevonden. Dit fenotype wordt veroorzaakt door een homozygote nonsense mutatie c.96T>A (p.(Cys32X)) in CLIC5. In dit hoofdstuk wordt ook aangetoond dat CLIC5 in verschillende menselijke weefsels tot expressie komt. Naast de milde nierfunctie stoornis werden er geen andere afwijkingen gevonden. In 213 patiënten van voornamelijk Nederlandse en Spaanse afkomst met een verdenking op een autosomaal recessief overervend niet-syndromaal gehoorverlies werd mutatieanalyse voor CLIC5 uitgevoerd. Hierbij werden geen nieuwe pathogene varianten gevonden. De conclusie in dit hoofdstuk is dan ook dat CLIC5 een nieuw doofheidsgen is, maar dat mutaties in dit gen geen frequente oorzaak zijn voor slechthorendheid in de Nederlandse of Spaanse populatie. Hoofdstuk 3 is een onderzoek met doel het fenotype van DFNB1, de meest voorkomende vorm van erfelijke slechthorendheid, uit te breiden. Het audiologische fenotype is zeer heterogeen en eerder uitgebreid onderzocht en beschreven. Over bijpassende anomalieën van het os temporale of de vestibulaire functie is minder bekend en in de literatuur is er over deze onderwerpen voor DFNB1 dan ook geen consensus. In dit hoofdstuk werden de vestibulaire functie en/of de resultaten van beeldvorming van het rotsbeen bij een grote groep van 44 DFNB1 patiënten retrospectief geëvalueerd. Vijf patiënten rapporteerden evenwichtsproblemen of lieten een afwijkende motorische ontwikkeling zien. Tijdens het draaistoelonderzoek lieten alle onderzochte patiënten echter een respons zien. Van de patienten had 65% een respons binnen de normaalwaarden bij calorisatie van beide oren en de ‘video head impulse’ test was normaal in alle geteste patiënten. Vestibulaire disfunctie is dan ook niet gecorreleerd aan het DFNB1 fenotype. Aanbevolen wordt om de sacculaire en utriculaire functie van het vestibulum nader te onderzoeken Summary / Samenvatting | 205 omdat dit in deze studie niet is onderzocht en er in de literatuur aanwijzingen zijn dat het connexine 26 eiwit hier mogelijk wel een belangrijke rol speelt. In 34,4% van de CT scans werden één of meer afwijkingen gezien van het rotsbeen. Deze anomalieën waren zeer variabel en kwamen elk maar in kleine aantallen patiënten voor. Geen enkele anomalie kan dan ook overtuigend gecorreleerd worden aan het DFNB1 genotype. In hoofdstuk 4 ligt de focus niet op het gehoorverlies, maar op de impact van een genetische diagnose van erfelijk gehoorverlies. Achtenveertig patiënten werden gevraagd om de volgende vragenlijsten: ‘Hospital Anxiety Depression Scale’, ‘Impact of Event Scale’, ‘Self Efficacy 24’, ‘Illness Cognition Questionnaire’ en de ‘Inventory for Social Reliance’ in te vullen. Algemene kenmerken werden verzameld door een samengestelde vragenlijst en statusonderzoek te gebruiken. De vragenlijsten dienden op drie momenten te worden ingevuld: ten tijde van de aanvraag van genetisch onderzoek, direct na de uitslag van het genetisch onderzoek en zes weken nadien. Tussen de patiënten die een genetische verklaring kregen voor hun gehoorverlies (positief resultaat) en de patiënten die dat niet kregen (negatief resultaat) werden geen significante verschillen gevonden. Dit onderzoek bevestigt ook dat een aantal slechthorenden die vragen over de oorzaak van hun slechthorendheid hebben, zich kunnen presenteren met een hoge mate van psychologische stress. Daarom wordt geadviseerd om extra aandacht te besteden aan het psychologisch welzijn van slechthorenden die zich met vragen naar de oorzaak van hun gehoorverlies presenteren. Genetische diagnostiek naar de oorzaak van de slechthorendheid heeft echter geen positief noch negatief effect op dit welzijn. Derhalve zijn er geen redenen om genetische diagnostiek naar erfelijke slechthorendheid wel of niet te verrichten. Dit proefschrift draagt bij aan de toename van kennis over autosomaal recessief overervend gehoorverlies. Dit is hier echter maar een klein onderdeel van en uitgebreider onderzoek is dan ook essentieel om patiënten die de oorzaak van hun slechthorendheid willen weten beter voor te kunnen lichten. De toekomstige ontwikkeling van nieuwe technieken om makkelijker genen of mutaties te identificeren, de cochlea in meer detail te kunnen onderzoeken en mogelijkheden om erfelijke slechthorendheid te kunnen behandelen, zullen leiden tot het uiteindelijke doel: een normaal gehoor 7 Dankwoord Dankwoord | 209 Dankwoord Het dankwoord, veelal het meest gelezen hoofdstuk van een proefschrift. Ook ik heb hier uitermate mijn best op gedaan, want zonder de hulp van velen had dit boekje er nu niet gelegen. Allereerst hartelijk dank aan alle patiënten en familieleden die hebben meegewerkt aan dit onderzoek. Dank dat ik jullie medische gegevens mocht gebruiken en dat jullie de tijd hebben genomen om al die vragenlijsten in te vullen. Mijn co-promotor, Dr. Pennings, beste Ronald, de beste co-promotor die ik me kon wensen. Jouw tomeloze enthousiasme heeft ervoor gezorgd dat dit proefschrift er nu ligt. Avonden, weekenden, vakanties, je was altijd bereikbaar. En als ik het even niet meer zag zitten wist jij mij altijd weer te motiveren om door te gaan. Dank voor alle tijd, betrokkenheid en energie. Geachte professor Kremer, beste Hannie. Dank voor de introductie in de moleculaire genetica en de mogelijkheid om ervaring op te doen in het laboratorium. Jouw kritische blik heeft dit proefschrift naar een hoger niveau getild. Beste leden van de manuscript commissie; professor Cremers, professor van Dijk en doctor Topsakal. Hartelijk dank voor het lezen en beoordelen van mijn proefschrift. Geachte professor Snik, beste Ad, hartelijk dank voor het meedenken op het vlak van de psychophysica. Heel fijn hoe jij dit inzichtelijk wist te maken. Beste Joop Leijendeckers, altijd bereid om mee te denken, altijd bereid om nog een meting te verrichten. Dank voor de goede samenwerking. Beste medewerkers van het audiologisch centrum, dank voor alle metingen die jullie voor dit proefschrift verricht hebben. Beste Patrick Huygen, dank voor de hulp bij de statistiek en je geduld om dit (soms meerdere malen) uit te leggen. Beste leden van de otogenetica werkgroep, veel over de otogenetica heb ik van jullie mogen leren. Daarnaast waren jullie altijd bereid om mee te denken, dank daarvoor! 210 | Chapter 7 Medewerkers van de afdeling vestibulogie, dank voor al jullie metingen. Andy, hartelijk dank voor al de uitleg en inzet. Jouw begeleiding was onmisbaar, vooral voor hoofdstuk 3. Een half jaar lang heb ik op het laboratorium gepoogd om de oorzaak achter de slechthorendheid in verschillende families te achterhalen. Zo’n dokter op het lab was soms wel even wennen, maar uiteindelijk heb ik hier veel geleerd. Dank aan alle medewerkers voor de behulpzaamheid. In het bijzonder dank aan Jaap: jouw geduld en manier van uitleggen zijn bewonderenswaardig. Dank dat je me dat half jaar wilde begeleiden. Beste Margit, ook jij hartelijk dank voor de begeleiding. Beste Erwin, jouw enthousiasme is eindeloos. Door met jou te discussiëren over het onderzoek werd mijn motivatie voor dit proefschrift altijd weer aangewakkerd. Dank daarvoor! Beste Lisette, helaas hebben we ons PTPRQ project niet samen kunnen afronden, maar gezellig was het altijd zeker. Heel fijn om gewoon even gezellig te kunnen kletsen, dank daarvoor! Beste Ramon, dank voor de samenwerking. Heel leuk om tijdens dit promotietraject ook een klein uitstapje naar de oogheelkunde te maken. Beste staf van de afdeling keel-, neus- en oorheelkunde van het Radboud UMC, dank voor het in mij gestelde vertrouwen en de kans om mijn opleiding bij jullie te volgen en te combineren met dit promotietraject. Beste dames van het stafsecretariaat, dank voor jullie hulp bij al mijn logistieke vragen. In het speciaal dank ik Loes Temmink, jij wist altijd de moeilijkste agenda puzzel op te lossen maar was ook altijd in om even gezellig te kletsen. Beste (oud) arts-assistenten, researchers en PA: Godelieve, Stijn, Hans, AnneMartine, Veronique, Rabia, Arthur, Maarten, Richard, Hubert, Lisette, Erik, Annemarie, Caroline, Henrieke, Jasmijn, Ingrid, Eline, Ruud, Rik, Saskia, Thijs, Josephine, Charlotte, Corinne, Chrisje, Luuk, Bas, Mieke, Machteld, Mayke, Ivo en David. Voordat ik ging solliciteren was ik nog nooit in Nijmegen geweest, maar onze groep maakte dat ik me hier snel helemaal thuis voelde. Dank voor alle gezelligheid binnen en buiten het Radboud! Union Dames 4, wat ben ik blij dat ik met jullie mag hockeyen. Twee keer per week de gelegenheid om alles eruit te lopen en te slaan. Vanaf nu ga ik ook weer voor een basisplek in de derde helft! Dankwoord | 211 Lieve clubgenoten, al 10 jaar samen door dik en dun. Dank voor de nodige afleiding die jullie hebben gegeven afgelopen jaren. Lieve Teun en Joris, mijn grote kleine broertjes. Dank voor de nodige afleiding tijdens dit proefschrift in de vorm van gekke foto’s over de app of een borrel drinken op Schiermonnikoog, de volgende keer laat ik mijn laptop thuis! Lieve Ingrid, jouw ambitie en doorzettingsvermogen hebben afgelopen jaren zeker geïnspireerd. Maar daarnaast breng je vooral veel gezelligheid en afleiding in fietsen, eten, lachen! Fijn dat je als medegroninger/ LvBer naast me wilt staan. Lieve Charlotte, grote zus, rond dezelfde tijd zijn we begonnen aan een ‘vervolg’ van onze opleiding. Jij hebt het inmiddels afgerond. Nu ik nog! Wat fijn dat je me daarbij wilt ondersteunen als paranimf. En Jeroen, fijn om te weten dat jij daar met jouw rust achter staat. Lieve Han en Sieka, dank voor jullie interesse en support. Ik ben blij dat we jullie nu zo dicht in de buurt hebben. Lieve Loes, heel fijn hoe jij altijd luistert en zo nodig relativeert. Onze dagjes uit waren altijd een hele fijne afleiding. Beste Sjoerd, dat jij probeerde om mij op twee jarige leeftijd desoxyribonucleinezuur te laten zeggen zou wel eens de basis voor dit proefschrift kunnen zijn geweest. Met z’n drieën hebben we heel wat uren mijn opleiding en onderzoek doorgenomen, dank dat jullie er zijn! Lieve papa en mama, van jullie mocht ik altijd worden wat ik maar wilde. Jullie hebben me de basis gegeven om uiteindelijk te promoveren en de opleiding tot KNO-arts te volgen en me daarbij zo goed mogelijk ondersteund. En als de energie dan echt op was, was het goed bijkomen in het Hoge Noorden. Dank dat jullie er voor me zijn. Liefste Ernst, jij bent altijd onvoorwaardelijk in mij blijven geloven, ook al had ik het al lang opgegeven. Zonder jouw liefde en steun was het me nooit gelukt. Hoe vaak hebben we niet gezegd: als het straks af is.. En die straks is nu. Ik kijk uit naar de toekomst met jou! 8 Curriculum Vitae Curriculum Vitae | 215 Curriculum Vitae Anne Oonk werd op 3 mei 1987 geboren te Haarlem. Zij groeide op in Hoofddorp met haar ouders, zus en twee broertjes. In 2000 verhuisden zij naar Dokkum, alwaar Anne in 2005 haar eindexamen behaalde aan het VWO van het Dockinga College. Hierna startte zij met haar studie Geneeskunde aan de Rijksuniversiteit Groningen. Naast haar studie was zij onder andere actief bij de lustrumcommissie van M.F.V. Panacea, de public relations committee van het International Student Congress of (Bio)Medical Sciences (ISCOMS) en op het hockeyveld van GCHC. In 2008 volgde zij een klinische stage op de kinderafdeling van het El Mansoura University Hospital in El Mansoura, Egypte. Tijdens het reguliere KNO coschap in het Deventer Ziekenhuis werd de interesse voor de Keel-, Neus- en Oorheelkunde gewekt. De reguliere coschappen werden afgesloten met een coschap sociale geneeskunde in het Ngwelezana Hospital in Empageni, Zuid Afrika. In 2011 startte zij met haar onderzoek naar triple negatieve borstkanker tumoren in het Deventer Ziekenhuis in samenwerking met het Antoni van Leeuwenhoek in Amsterdam. Hierop volgde een keuze coschap KNO in het UMC in Utrecht en in het Deventer Ziekenhuis. Eind 2011 behaalde zij haar artsenexamen en startte zij als artsonderzoeker aan het Radboud UMC onder begeleiding van Dr. R.J.E. Pennings en Prof. Dr. H. Kremer. Dit onderzoekstraject resulteerde uiteindelijk in dit proefschrift. Sinds 2013 is zij in opleiding tot KNO-arts in het Radboud UMC onder Prof. Dr. H.A.M. Marres en opleider Dr. F.J.A. van den Hoogen. 9 List of publications List of publications | 219 List of publications E.H. Lips, N. Laddach, S.P. Savola, M.A. Vollebergh, A.M.M. Oonk, A.L.T. Imholz, L.F. Wessels, J. Wesseling, P.M. Nederlof and S. Rodenhuis (2011).”Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness.” Breast Cancer Res 13(5): R107. A.M.M. Oonk, C. van Rijn, M.M. Smits, L. Mulder, N. Laddach, S.P. Savola, J. Wesseling, S. Rodenhuis, A.L.T Imholz and E.H. Lips (2012).”Clinical correlates of ‘BRCAness’ in triple-negative breast cancer of patients receiving adjuvant chemotherapy.” Ann Oncol 23(9): 2301-2305. K.O. Yariz, D. Duman, C.Z. Seco, J. Dallman, M. Huang, T.A. Peters, A. Sirmaci, N. Lu, M. Schraders, I. Skromne, J. Oostrik, O. Diaz-Horta, J.I. Young, S. Tokgoz-Yilmaz, O. Konukseven, H. Shahin, L. Hetterschijt, M. Kanaan, A.M.M. Oonk, Y.J. Edwards, H. Li, S. Atalay, S. Blanton, A.A. Desmidt, X.Z. Liu, R.J.E. Pennings, Z. Lu, Z.Y. Chen, H. Kremer and M. Tekin (2012).”Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss.” Am J Hum Genet 91(5): 872-882. M. Schraders, L. Ruiz-Palmero, E. Kalay, J. Oostrik, F.J. del Castillo, O. Sezgin, A.J. Beynon, T.M. Strom, R.J.E. Pennings, C.Z. Seco, A.M.M. Oonk, H.P.M. Kunst, M. Dominguez-Ruiz, A.M. Garcia-Arumi, M. del Campo, M. Villamar, L.H. Hoefsloot, F. Moreno, R.J.C. Admiraal, I. del Castillo and H. Kremer (2012).”Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment.” Am J Hum Genet 91(5): 883-889. E.H. Lips, L. Mulder, A.M.M. Oonk, L.E. van der Kolk, F.B. Hogervorst, A.L.T. Imholz, J. Wesseling, S. Rodenhuis and P.M. Nederlof (2013).”Triple-negative breast cancer: BRCAness and concordance of clinical features with BRCA1-mutation carriers.” Br J Cancer 108(10): 2172-2177. A.M.M. Oonk, J.M. Leijendeckers, E.M. Lammers, N.J. Weegerink, J. Oostrik, A.J. Beynon, P.L.M. Huygen, H.P.M. Kunst, H. Kremer, A.F.M. Snik and R.J.E. Pennings (2013).”Progressive hereditary hearing impairment caused by a MYO6 mutation resembles presbyacusis.” Hear Res 299: 88-98. 220 | Chapter 9 D. Ragancokova, E. Rocca, A.M.M. Oonk, H. Schulz, E. Rohde, J. Bednarsch, I. Feenstra, R.J.E. Pennings, H. Wende and A.N. Garratt (2014).”TSHZ1-dependent gene regulation is essential for olfactory bulb development and olfaction.” J Clin Invest 124(3): 1214-1227. A.M.M. Oonk, M.S. Ekker, P.L.M. Huygen, H.P.M. Kunst, H. Kremer, J.J. Schelhaas and R.J.E. Pennings (2014).”Intrafamilial variable hearing loss in TRPV4 induced spinal muscular atrophy.” Ann Otol Rhinol Laryngol 123(12): 859-865. A.M.M. Oonk, J.M. Leijendeckers, P.L.M. Huygen, M. Schraders, M. del Campo, I. del Castillo, M. Tekin, I. Feenstra, A.J. Beynon, H.P.M. Kunst, A.F.M. Snik, H. Kremer, R.J.C. Admiraal and R.J.E. Pennings (2014).”Similar phenotypes caused by mutations in OTOG and OTOGL.” Ear Hear 35(3): e84-91. A.M.M. Oonk, S.C. Steens and R.J.E. Pennings (2014).”Radiologic confirmation of patulous eustachian tube by recumbent computed tomography.” Otol Neurotol 35(3): e117-118. A.M.M. Oonk, R.A.C. van Huet, J.M. Leijendeckers, J. Oostrik, H. Venselaar, E. van Wijk, A.J. Beynon, H.P.M. Kunst, C.B. Hoyng, H. Kremer, M. Schraders and R.J.E. Pennings (2015).”Nonsyndromic hearing loss caused by USH1G mutations: widening the USH1G disease spectrum.” Ear Hear 36(2): 205-211. C.Z. Seco, A.M.M. Oonk, M. Dominguez-Ruiz, J.M.T. Draaisma, M. Gandia, J. Oostrik, K. Neveling, H.P.M. Kunst, L.H. Hoefsloot, I. del Castillo, R.J.E. Pennings, H. Kremer, R.J.C. Admiraal and M. Schraders (2015).”Progressive hearing loss and vestibular dysfunction caused by a homozygous nonsense mutation in CLIC5.” Eur J Hum Genet 23(2): 189-194. C.Z. Seco, A.P. Giese, S. Shafique, M. Schraders, A.M.M. Oonk, M. Grossheim, J. Oostrik, T. Strom, R. Hegde, E. van Wijk, G.I. Frolenkov, M. Azam, H.G. Yntema, R.H. Free, S. Riazuddin, J.B. Verheij, R.J.C. Admiraal, R. Qamar, Z.M. Ahmed and H. Kremer (2015).”Novel and recurrent CIB2 variants, associated with nonsyndromic deafness, do not affect calcium buffering and localization in hair cells.” Eur J Hum Genet (Epub ahead of print). A.M.M. Oonk, A.J. Beynon, T.A. Peters, H.P.M. Kunst, R.J.C. Admiraal, H. Kremer, B. Verbist and R.J.E. Pennings (2015).”Vestibular function and temporal bone imaging in DFNB1.” Hear Res 327:227-34. List of publications | 221 E. van Beelen, A.M.M. Oonk, J.M. Leijendeckers, R.J.E. Pennings, H.J. Dieker, A.F.M. Snik, H. Kremer, H.P.M. Kunst (2015).“Audiometric Characteristics of a Dutch DFNA10 Family With Mid-Frequency Hearing Impairment.”Hear Res (Epub ahead of print). A.M.M. Oonk, P.L.M. Huygen, H.P.M. Kunst, H. Kremer, R.J.E. Pennings (2015). Features of autosomal recessive nonsyndromic hearing impairment; a review to serve as a reference. Clin Otol (Epub ahead of print). 10 List of abbreviations List of abbreviations | 225 List of abbreviations ABR: ANSD: arNSHI: ARTA: ATD: BCVA: CI: CMV: CNC: CT: cVEMPS: dB HL: Del: DFNA: DFNB: DFN: DLF: EAC: EAS: EBV: EF: ERG: EVA: EVS: FAF: FM: GPRI: HADS: HFASCs: HRR: HSCC: ICQ: IES: IQR: ISCED: ISCEV: ISR: Jbg: auditory-evoked brainstem response auditory neuropathy spectrum disorder autosomal recessive non-syndromic hearing impairment age related typical audiograms annual threshold deterioration best-corrected visual acuity cochlear implant cytomegalo virus cochlear nerve canal computed tomography cervical vestibular-evoked myogenic potentials decibel hearing level deletion autosomal dominant inherited deafness autosomal recessive inherited deafness deafness difference limen for frequency external auditory canal electro acoustic stimulation Epstein Barr Virus endolymphatic fossa electroretinography enlarged vestibular aqueduct exome variant server fundus autofluorescence frequency modulation genetic psychological risk inventory hospital anxiety and depression scale human fetal auditory stem cells Hardy-Rand-Rittler horizontal semicircular canal illness cognition questionnaire impact of event scale interquartile range international standard classification of education international society for clinical electrophysiology of vision inventory of social reliance Jitterbug 226 | Chapter 10 kHz: MCL: MET: ms: MRI: NMD: n.s.: OAEs: OCT: Pa: PTA: qPCR: RP: RPE: SCC: SD-OCT: SE24: SNP: S/N: SPL: SRT: SSCC: STR: VA: VEMPs: vHIT: VNTR: VST: WES: yr: kilo Hertz most comfortable listening level mechanoelectric transductor millisecond magnetic resonance imaging nonsense-mediated mRNA decay not significant otoacoustic emissions optical coherence tomography Pascal pure tone audiometry quantitative polymerase chain reaction retinitis pigmentosa retinal pigment epithelium semicircular canal spectral-domain optical coherence tomography self efficacy scale single nucleotide polymorphism signal/ noise sound pressure level speech reception threshold superior semicircular canal single tandem repeat vestibular aqueduct vestibular-evoked myogenic potentials video head impulse test variable number tandem repeat velocity step test whole exome sequencing year